Multiphoton Direct Laser Writing and 3D Imaging of Polymeric Freestanding Architectures for Cell Colonization

Abstract

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 fabrication of 3D freestanding scaffolds, realized by multiphoton direct laser writing and seeded with neuroblastoma cells, and their multitechnique characterization using advanced 3D fluorescence imaging approaches. The high accuracy of the fabrication process (≈200 nm) allows a much finer control of the micro- and nanoscale features compared to other 3D printing technologies based on fused deposition modeling, inkjet printing, selective laser sintering, or polyjet technology. Scanning electron microscopy (SEM) provides detailed insights about the morphology of both cells and cellular 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, is unveiled by light sheet fluorescence microscopy and multiphoton confocal imaging highlighting 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.

Engineered 3D Polymer and Hydrogel Microenvironments for Cell Culture Applications

“…Two-photon polymerization (TPP) uses the non-linearity of two-photon absorption to polymerize extremely confined regions (voxels have a characteristic dimension in the order of 100 nm) of resin since only the focal spot receives sufficient light intensity for triggering the two-photon process. TPP has much higher 3D resolution than SLA but the writing time is much slower, making it suitable for special applications and rapid prototyping but not for high throughput production [60][61][62][63], although the integration of galvanometric mirrors and moving-beam fixed-sample (MBFS) strategies are starting to fill this gap.…”

Nanoscale investigation in 3D scaffolds of cell-material interactions for tissue-engineering

“…Here, particular attention has been dedicated to the development of 3D systems that better replicate the structural organization of living tissues and that, compared to 2D platforms, have been extensively demonstrated to affect cellular behavior in a more realistic way 11,12 . For instance, 3D matrices/scaffolds have been fabricated with different degrees of structural organization to correlate in vitro cells responses with different and well-defined cell-material interactions [13][14][15][16] . In this context, in order to evaluate the effect of specific functional features upon cellular response, one can get fundamental informative cues from the investigation of the interface between cells and 3D biomaterials at the macro, micro and nanoscales.…”

“…Depending on the scaffolds architecture, cells can partially adhere to the local surface area or even be suspended across the pore bridging two sites of the support material. These conditions reflect high tensional states of the membrane which could often result in cracks when specimens are prepared with hard drying techniques 16 . This aspect limits the possibility to fully characterize cellmaterial interactions in complex cellular 3D systems and then the capability to properly evaluate the effect of a peculiar design on cell adhesion processes.…”

“…The developed procedure allowed us to access and characterize cells positioned in locations inaccessible with other approaches 16 . Here, considering a top view of an ordered scaffold with cells, we were indeed able to analyze 3 different relative positions of adherent cells in respect to the arms of the ordered scaffold: cells adherent to the strut surface (Figure 3A), cells suspended between two struts (Figure 3C), cells partially attaching to one arm (Figure 3E).…”

“…In this case, cells freely colonize the scaffold surface and penetrate in the pores, depositing an abundant layer of ECM (Figure 4), necessary for cell adhesion, survival and cell to cell communication as well as to adapt to the culture substrate properties 32,35 . Compared to the case of ordered scaffold it is worth noting that in this case, because of both the irregular scaffold architecture and the complex cellular rearrangement, the most advanced microscopy techniques find greater limitations in imaging the scaffold core 16 . While for ordered scaffolds it is possible to create a cross section following the geometry of the 3D structure and the relative position of cells on it, in the case of non-ordered scaffolds the region of interest is located mainly based on the upmost layer of cell visible via SEM ( Figure 4A).…”