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Glioblastoma (GBM) is a devastating cancer of the brain with an extremely poor prognosis. For this reason, besides clinical and preclinical studies, novel in vitro models for the assessment of cancer response to drugs and radiation are being developed. In such context, three-dimensional (3D)engineered cellular microenvironments, compared to unrealistic two-dimensional (2D) monolayer cell culture, provide a model closer to the in vivo configuration. Concerning cancer treatment, while X-ray radiotherapy and chemotherapy remain the current standard, proton beam therapy is an appealing alternative as protons can be efficiently targeted to destroy cancer cells while sparing the surrounding healthy tissue. However, despite the treatment's compelling biological and medical rationale, little is known about the effects of protons on GBM at the cellular level. In this work, we designed novel 3D-engineered scaffolds inspired by the geometry of brain blood vessels, which cover a vital role in the colonization mechanisms of GBM cells. The architectures were fabricated by two-photon polymerization (2PP), cultured with U-251 GBM cells and integrated for the first time in the context of proton radiation experiments to assess their response to treatment. We employed Gamma H2A.X as a fluorescent biomarker to identify the DNA damage induced in the cells by proton beams. The results show a higher DNA doublestrand breakage in 2D cell monolayers as compared to cells cultured in 3D. The discrepancy in terms of proton radiation response could indicate a difference in the radioresistance of the GBM cells or in the rate of repair kinetics between 2D cell monolayers and 3D cell networks. Thus, these biomimetic-engineered 3D scaffolds pave the way for the realization of a benchmark tool that can be used to routinely assess the effects of proton therapy on 3D GBM cell networks and other types of cancer cells. KEYWORDS: engineered cell microenvironments, two-photon polymerization, cancer, glioblastoma, proton therapy, DNA damage

Surface-enhanced Raman scattering (SERS) substrates are of great interest for detecting low-concentrated analytes. However, issues such as multistep processing, cost, and possible presence of hazardous substances in the fabrication still represent a significant drawback. In this paper, an innovative direct writing method is introduced for solvent-free and spatially selective deposition of fine metal copper nanoparticles (CuNPs), with size distribution below 20 nm, generated in-line through a spark ablation method (SAM). The deposited CuNPs' morphology and composition were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), and energy-dispersive X-ray spectroscopy (EDS). The resulting CuNP patterns feature porous 3D microdomains with nanometric structures serving as hot spots for Raman signal enhancement. Low-temperature post-treatment (below 200 °C) of the deposited CuNPs significantly evolves its morphology and leads to sintering of NPs into a semicrystalline structure with sharp geometric features, which resulted in a more than 10-fold increase of the enhancement factor (up to 2.1 × 105) compared to non-heat-treated samples. The proposed method allows creating SERS substrates constituted by sharp 3D metallic nanopatterns selectively deposited onto specific regions, which paves the way for new printed, highly sensitive SERS-based sensors.

Biomimicking biological niches of healthy tissues or tumors can be achieved by means of artificial microenvironments, where structural and mechanical properties are crucial parameters to promote tissue formation and recreate natural conditions. In this work, three-dimensional (3D) scaffolds based on woodpile structures were fabricated by two-photon polymerization (2PP) of different photosensitive polymers (IP-S and SZ2080) and hydrogels (PEGDA 700) using two different 2PP setups, a commercial one and a customized one. The structures' properties were tuned to study the effect of scaffold dimensions (gap size) and their mechanical properties on the adhesion and proliferation of bone marrow mesenchymal stem cells (BM-MSCs), which can serve as a model for leukemic diseases, among other hematological applications. The woodpile structures feature gap sizes of 25, 50, and 100 μm and a fixed beam diameter of 25 μm, to systematically study the optimal cell colonization that promotes healthy cell growth and potential tissue formation. The characterization of the scaffolds involved scanning electron microscopy and mechanical nanoindenting, while their suitability for supporting cell growth was evaluated with live/dead cell assays and multistaining 3D confocal imaging. In the mechanical assays of the hydrogel material, we observed two different stiffness ranges depending on the indentation depth. Larger gap woodpile structures coated with fibronectin were identified as the most promising scaffolds for 3D BM-MSC cellular models, showing higher proliferation rates. The results indicate that both the design and the employed materials are suitable for further assays, where retaining the BM-MSC stemness and original features is crucial, including studies focused on BM disorders such as leukemia and others. Moreover, the combination of 3D scaffold geometry and materials holds great potential for the investigation of cellular behaviors in a co-culture setting, for example, mesenchymal and hematopoietic stem cells, to be further applied in medical research and pharmacological studies.

One of the essential requirements to create nanoparticle (NP)-based applications and functions is the ability to control their deposition in specific locations. Many methods have been proposed, with wet direct writing (DW) techniques such as inkjet printing being the most employed. These methods generally depend on off-line and solvent-based NP synthesis leading to contamination and impurity in the final NP film as well as inhomogeneity in the deposition caused by solution-substrate interactions. This paper introduces a dry aerosol direct writing (dADW) method, which combines spark ablation-based and solvent-free NP synthesis with spatially selective deposition using aerodynamic focusing in a vacuum chamber. The challenge is to print high-resolution lines and spots of nanoparticles with a diameter < 100 nm. We study two aerodynamic nozzle concepts, a converging nozzle (CN) and a sheath gas nozzle (SGN), and investigate numerically how their design, as well as operating parameters, relate to the deposition process performance. This is quantified by three criteria: contraction factor, focusing ratio, and collection efficiency. We also compared our numerical results to experimental assays by manufacturing two SGNs and three CNs and evaluating the performance of each nozzle in terms of resolution, sharpness and thickness of the line. Using one of the SGN designs with an outlet diameter of 248 µm and an aerosol to total flow rate ratio of 0.17, we achieved a high-resolution line with a width of 67 µm, i.e., equal to 27% of the nozzle diameter, when printing < 100 nm Au NPs. The presented additive manufacturing method enables, therefore, the creation of high-resolution and sharp patterns of metallic nanoparticles, which can be employed in a wide range of applications, ranging from interconnects to optical and gas sensors.

International audience; The design of advanced functional devices often requires the use of intrinsically curved geometries that belong to the realm of non-Euclidean geometry and remain a challenge for traditional engineering approaches. Here, it is shown how the simple deflection of thick meta-plates based on hexagonal cellular mesostructures can be used to achieve a wide range of intrinsic (i.e., Gaussian) curvatures, including dome-like and saddle-like shapes. Depending on the unit cell structure, non-auxetic (i.e., positive Poisson ratio) or auxetic (i.e., negative Poisson ratio) plates can be obtained, leading to a negative or positive value of the Gaussian curvature upon bending, respectively. It is found that bending such meta-plates along their longitudinal direction induces a curvature along their transverse direction. Experimentally and numerically, it is shown how the amplitude of this induced curvature is related to the longitudinal bending and the geometry of the meta-plate. The approach proposed here constitutes a general route for the rational design of advanced functional devices with intrinsically curved geometries. To demonstrate the merits of this approach, a scaling relationship is presented, and its validity is demonstrated by applying it to 3D-printed microscale meta-plates. Several applications for adaptive optical devices with adjustable focal length and soft wearable robotics are presented.

International audience; The development of advanced techniques of fabrication of three-dimensional (3D) microenvironments for the study of cell growth and proliferation has become one of the major motivations of material scientists and bioengineers in the past decade. Here, we present a novel residueless 3D structuration technique of poly(dimethylsiloxane) (PDMS) by water-in-PDMS emulsion casting and subsequent curing process in temperature-/pressure-controlled environment. Scanning electron microscopy and X-ray microcomputed tomography allowed us to investigate the impact of those parameters on the microarchitecture of the porous structure. We demonstrated that the optimized emulsion casting process gives rise to large-scale and highly interconnected network with pore size ranging from 500μm to 1.5 mm that turned out to be nicely adapted to 3D cell culture. Experimental cell culture validations were performed using SaOS-2 (osteosarcoma) cell lines. Epifluorescence and deep penetration imaging techniques as two-photon confocal microscopy unveiled information about cell morphology and confirmed a homogeneous cell proliferation and spatial distribution in the 3D porous structure within an available volume larger than 1 cm3. These results open alternative scenarios for the fabrication and integration of porous scaffolds for the development21of 3D cell culture platforms.

International audience; The realization of 2D and 3D meso-scale architectures is an area of research involving a wide range of disciplines ranging from materials science, microelectronics, phononics, microfluidics to biomedicine requiring millimeter to centimeter-sized objects embedding micrometric features. In the recent years, several technologies have been employed to provide optimal features in terms of object size flexibility, printing resolution, large materials library and fabrication speed. In this work, we report a fully customizable single-photon absorption 3D fabrication methodology based on direct laser fabrication. To validate this approach and highlight the versatility of the setup, we have fabricated a comprehensive ensemble of 2D and 3D designs with potential applications in biomimetics, 3D scaffolding and microfluidics. The high degree of tunability of the reported fabrication system allows tailoring the laser power, slicing and fabrication speed for each single area of the design. These unique features enable a rapid prototyping of millimeter to centimeter-sized objects involving 3D architectures with true freestanding subunits and micrometric feature reproducibility. The presented strategy fills indeed the current technological gap related to the development of meso-scale architectures required in multidisciplinary fields of research.

International audience; In this work, we report on the adhesion of HCT116 (human colon carcinoma cells) cultured on nanofibrillar polymethylmethacrylate (PMMA) and SU-8 micropillars substrates. Both surfaces enabled a good cell proliferation and promoted the formation of adherent interconnections with the fabricated nano- and microstructures. The three-dimensional immunofluorescence confocal characterization of the cells on nanotextured PMMA highlighted the expression of well-spread F-actin cytoskeletal networks as well as the presence of focal adhesions. This study provides thus interesting perspectives for further investigations on the force/adhesion mechanisms related to cancer cell growth and proliferation.

Amyloid β (Aβ) peptides are the main constituents of Alzheimer's amyloid plaques in the brain. Here we report how the unique microfluidic flows exerted by droplets sitting on superhydrophobic surfaces can influence the aggregation mechanisms of several Aβ fragments by boosting their fibril self-assembly. Aβ(25-35), Aβ(1-40), and Aβ(12-28) were dried both on flat hydrophilic surfaces (contact angle (CA) = 37.3°) and on nanostructured superhydrophobic ones (CA = 175.8°). By embedding nanoroughened surfaces on top of highly X-ray transparent Si3N4 membranes, it was possible to probe the solid residues by raster-scan synchrotron radiation X-ray microdiffraction (μXRD). As compared to residues obtained on flat Si3N4 membranes, a general enhancement of fibrillar material was detected for all Aβ fragments dried on superhydrophobic surfaces, with a particular emphasis on the shorter ones. Indeed, both Aβ(25-35) and Aβ(12-28) showed a marked crystalline cross-β phase with varying fiber textures. The homogeneous evaporation rate provided by these nanostructured supports, and the possibility to use transparent membranes, can open a wide range of in situ X-ray and spectroscopic characterizations of amyloidal peptides involved in neurodegenerative diseases and for the fabrication of amyloid-based nanodevices.

International audience; The realization of 3D architectures for the study of cell growth, proliferation and differentiation is a task of fundamental importance for both technological and biological communities involved in the development of biomimetic cell culture environments. Here we report the combination of 3D freestanding scaffolds realized by multi-photon direct laser writing (DLW), seeded with neuroblastoma cells, and their multi-technique characterization using advanced 3D fluorescence imaging techniques. The high accuracy of the fabrication process (≈ 200 nm) provides a much finer control of the meso-, micro-and nano-scale features compared to other 3D printing technologies based on fused deposition modeling, inkjet printing, selective laser sintering or polyjet technology. Scanning electron microscopy (SEM) provided detailed insights about the morphology of both cells and cellular Complete Manuscript interconnections around the 3D architecture. On the other hand, the nature of the seeding in the inner core of the 3D scaffold, inaccessible by conventional SEM imaging, was unveiled by light sheet fluorescence microscopy and multi-photon confocal imaging which highlighted an optimal cell colonization both around and within the 3D scaffold as well as the formation of long neuritic extensions. The results open appealing scenarios for the use of the developed 3D fabrication/3D imaging protocols in several neuroscientific contexts.

Here we are presenting a comparative analysis of conformational changes of two amyloid β peptides, Aβ(25-35) and Aβ(1-42), in the presence and absence of a phospholipid system, namely, POPC/POPS (1-palmitoyl-2-oleoylphospatidylcholine/palmitoyl-2-oleoylphospatidylserine), through Raman spectroscopy, synchrotron radiation micro Fourier-transform infrared spectroscopy, and micro X-ray diffraction. Ringlike samples were obtained from the evaporation of pure and mixed solutions of the proteins together with the POPC/POPS system on highly hydrophilic substrates. The results confirm the presence of a α-helical to β-sheet transition from the internal rim of the ringlike samples to the external one in the pure Aβ(25-35) residual, probably due to the convective flow inside the droplets sitting on highly hydrophilic substrates enhancing the local concentration of the peptide at the external edge of the dried drop. In contrast, the presence of POPC/POPS lipids in the peptide does not result in α-helical structures and introduces the presence of antiparallel β-sheet material together with parallel β-sheet structures and possible β-turns. As a control, Aβ(1-42) peptide was also tested and shows β-sheet conformations independently from the presence of the lipid system. The μXRD analysis further confirmed these conclusions, showing how the absence of the phospholipid system induces in the Aβ(25-35) a probable composite α/β material while its coexistence with the peptide leads to a not oriented β-sheet conformation. These results open interesting scenarios on the study of conformational changes of Aβ peptides and could help, with further investigations, to better clarify the role of enzymes and alternative lipid systems involved in the amyloidosis process of Aβ fragments.

Drops of exosome dispersions from healthy epithelial colon cell line and colorectal cancer cells were dried on a superhydrophobic PMMA substrate. The residues were studied by small- and wide-angle X-ray scattering using both a synchrotron radiation micrometric beam and a high-flux table-top X-ray source. Structural differences between healthy and cancerous cells were detected in the lamellar lattices of the exosome macro-aggregates.

The liquid/air interface of calcium bicarbonate solution drops was probed by synchrotron radiation microbeam scattering. The drops were deposited on a nanopatterned superhydrophobic poly(methyl methacrylate) surface and raster-scanned during evaporation by small-angle and wide-angle X-ray scattering. The appearance of about 200-nm-size calcite crystallites at the interface could be spatially resolved at the onset of crystallization. Diffuse scattering from the interface is attributed to a dense nanoscale amorphous calcium carbonate phase. Calcite was found to be the major phase in the solid residue with vaterite as minor phase.

Superhydrophobic poly(methyl methacrylate) surfaces with contact angles of ∼170° and high optical and X-ray transparencies have been fabricated through the use of optical lithography and plasma etching. The surfaces contain either a microscale pattern of micropillars or a random nanofibrillar pattern. Nanoscale asperities on top of the micropillars closely resemble Nelumbo nucifera lotus leaves. The evolution of the contact angle of water and lysozyme solution droplets during evaporation was studied on the micro- and nanopatterned surfaces, showing in particular contact-line pinning for the protein solution droplet on the nanopatterned surface. The microstructural evolution of lysozyme solution droplets was studied on both types of surfaces in situ under nearly contact-free conditions by synchrotron radiation microbeam wide-angle and small-angle X-ray scattering revealing the increasing protein concentration and the onset of precipitation. The solid residuals show hollow sphere morphologies. Rastermicrodiffraction of the detached residuals suggests about a 1/3 volume fraction of ≥17 nm lysozyme nanocrystalline domains and about a 2/3 short-range-order volume fraction. About 5-fold larger nanocrystalline domains were observed at the attachment points of the sphere to the substrates, which is attributed to particle growth in a shear flow. Such surfaces represent nearly contact-free sample supports for studies of inorganic and organic solution droplets, which find applications in biochips.