Cells in our bodies are arranged in three dimensions (3D) according to complex architectures, which play a fundamental role in the functionality of the living tissue. Nevertheless, nowadays most of the cell culture approaches still rely either on unrealistic 2-dimensional (2D) surfaces, unable to offer a 3D spatial configuration for cellular growth and proliferation, or hydrogel materials that, although providing 3D environments, often lack a fine control of their geometrical micro- and nano-scale features, which are crucial for guiding cell development and alignment of cellular interconnections.
In the field of tissue engineering, and in particular in the sub-branch of neuroscientific applications, one of the major issues is to have a support which from one side can ensure a sufficient level of adhesion and an optimal neuronal proliferation but, in view of further applications in regenerative medicine, needs ideal features such as biocompatibility, controlled biodegradability with non-toxic degradative products, porosity for vascularization, and cell migration. For these reasons, hydrogels [1] have emerged as promising materials as they can feature a high degree of tunability in terms of bio-functionalization and adjustable mechanical properties, as well as an extracellular cell matrix-like microenvironment for cell growth and tissue formation.
To date, the methodologies employed to manufacture 3D architectures for cell culture and tissue engineering applications [2] include, among the others, fused deposition modeling(FDM), ink-jet printing, laser-induced forward transfer (LIFT) and electrospinning that, although relying on a wide materials’ library, lack either the possibility to create true 3D free-standing architectures without the presence of sacrificial supports or cannot reach micrometric feature resolution. More recently, an increasing interest has been devoted toward light-assisted photopolymerization techniques [3], such as stereolithography (SLA), selective laser sintering (SLS) and digital light projection (DLP), where the 3D object is realized by exploiting a layer-by-layer approach from a series of transverse-plane image slices. These approaches can reach a few micrometer resolution although there is a more limited availability of materials capable to feature at the same time biocompatibility and photosensitive properties.
Another technique which is gaining popularity in the materials science community is Two-photon lithography direct laser writing (2PL-DLW) [4] that, compared to SLA approaches, can reach sub-micrometric feature resolution [5] but shares often the same limitation in terms of available biomaterials since most of the 2PL-DLW polymers are not formulated for biological applications due to the toxicity of many feedstock materials.
Here, we report the direct laser writing fabrication of Poly(ethylene glycol)diacrylate(PEGDA), a biocompatible hydrogel approved by the Food and Drug Administration (FDA), for supporting neuronal cell growth [6]. The 3D hydrogel cross porous woodpile architecture that features on the cover of this issue of Materials Today has been realized using 2PL-DLW. The physical mechanism lying behind the 2PL-DLW fabrication exploits the two-photon absorption (TPA) of near-infrared (NIR) radiation by focusing infrared femtosecond laser pulses onto an organic pre-polymer material highly absorptive in the UV radiation range while “transparent” in the IR one. This non-linear mechanism is tuned in order to induce the photopolymerization of the exposed material in extremely confined volumes called voxels whose dimension is mainly determined by the laser spot-size, the power of the laser sourceand the properties of the material itself. In order to make the PEGDA hydrogel sensitive to laser radiation, we mixed it with an appropriate photoinitiator (Phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide) highly absorptive in the UV range. Exploiting a galvanometric optical setup and a dip-in laser lithography configuration (DiLL), in which the microscope objective is directly immersed into the hydrogel solution and the focused laser moves directly within the photosensitive material, we show how it is possible to write 3D architectures with true overhanging structures featuring apertures along x, y, z axes. The ultra-fast writing speed provided by the galvanometric mirrors allowed to fabricate multiple architectures in very short time. After laser writing, the sample was immersed in de-ionized water to remove the unexposed PEGDA hydrogel without the use of other chemical solvents. The architecture was then functionalized with a combination of poly-L-lysine, to favor the electrostatic interactions between the negatively charged ions of the cellular membrane and the employed hydrogel, and laminin (a protein of the extracellular matrix, involved as well in the mechanisms of cell adhesion).
In order to assess the efficiency of the 3D PEGDA hydrogel scaffolds, we cultivated neuro2A cells, able to develop, under appropriate conditions, many properties of neurons, such as neuritic extensions. The low stiffness of PEGDA (Young’s modulus E?≈?200?kPa) represents a very appealing solution for the culture of neural cells, compared to the high stiffness (E?≈?2–3 GPa) of conventional materials employed in 2PL-DLW [7], as it is closer to brain stiffness (E?≈?600?Pa). Thanks to the porous nature of the proposed scaffold geometry, we observed the efficient growth of a ramified neuronal network throughout the 3D architecture highlighting the formation of multiple neuritic extensions per cell with a length between 10 and 60?μm.
In the framework of these investigations, conventional morphological characterization (based on Scanning Electron Microscopy) has been associated to advanced 3D fluorescence imaging techniques (Light Sheet Fluorescence Microscopy and Two-photon confocal imaging) in order to “shed light” on the localization and the morphology of the cells not only around the 3D scaffold but also within its most inaccessible core regions [5], [6]. In such context, PEGDA hydrogel showed very low intrinsic fluorescent emission (≈100 times lower than conventional polymeric materials employed in 2PL-DLW [7]), thus enabling a multi-staining immunofluorescence evaluation of the functional features of neuro2A cells in three dimensions. These unique “quasi-transparency” optical properties were crucial to allow a full 3D immunofluorescence reconstruction of the neuro2A colonization of the scaffold as well as the detection of F-Actin microfilaments and β-tubulin neuronal marker even “through” the PEGDA structures.
The reported 3D hydrogel architectures represent therefore both an appealing tool for further applications in neural tissue engineering and a dedicated 3D cellular microenvironment for the evaluation of biochemical compounds’ (e.g., drugs, neurodegenerative proteins [8]) influence on relevant 3D neural cell cultures.
Acknowledgments
The present work was supported by the LAAS-CNRS Carnot funding ‘PHANTOM 3D’, by the H2020 European project HOLIFab (Grant No. 760927) and by the LAAS-CNRS micro and nanotechnologies platform member of the French RENATECH network.
Further reading
[1] M. Verhulsel, et al.
Biomaterials, 35 (2014), pp. 1816-1832
[2] S.V. Murphy, A. Atala
Nat. Biotechnol., 32 (2014), pp. 773-785
[3] M. Guvendiren, et al.
ACS Biomater. Sci. Eng., 2 (2016), pp. 1679-1693
[4] A.K. Nguyen, R.J. Narayan
Mater. Today, 20 (2017), pp. 314-322
[5] A. Accardo, et al.
Small, 13 (2017), p. 1700621
[6] A. Accardo, et al.
Biomed. Phys. Eng. Express, 4 (2018), p. 027009
[7] L.R. Meza, et al.
Proc. Natl. Acad. Sci. U.S.A., 112 (2015), pp. 11502-11507
[8] A. Accardo, et al.
ACS Appl. Mater. Interfaces, 7 (2015), pp. 20875-20884