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Three-dimensional cancer cell cultures have been a valuable research model for developing new drug targets in the preclinical stage. However, there are still limitations to these in vitro models. Scaffold-based systems offer a promising approach to overcoming these challenges in cancer research. In this study, we show that two-photon polymerization (TPP)-assisted printing of scaffolds enhances 3D tumor cell culture formation without additional modifications. TPP is a perfect fit for this task, as it is an advanced 3D-printing technique combining a μm-level resolution with complete freedom in the design of the final structure. Additionally, it can use a wide array of materials, including biocompatible ones. We exploit these capabilities to fabricate scaffolds from two different biocompatible materials-PEGDA and OrmoClear. Cubic spheroid scaffolds with a more complex architecture were produced and tested. The biological evaluation showed that the human ovarian cancer cell lines SKOV3 and A2780 formed 3D cultures on printed scaffolds without a preference for the material. The gene expression evaluation showed that the A2780 cell line exhibited substantial changes in CDH1 , CDH2 , TWIST , COL1A1 , and SMAD3 gene expression, while the SKOV3 cell line had slight changes in said gene expression. Our findings show how the scaffold architecture design impacts tumor cell culture 3D spheroid formation, especially for the A2780 cancer cell line.

Organ-on-a-chip devices are gaining popularity in medical research due to the possibility of performing extremely complex living-body-resembling research in vitro. For this reason, there is a substantial drive in developing technologies capable of producing such structures in a simple and, at the same time, flexible manner. One of the primary challenges in producing organ-on-chip devices from a manufacturing standpoint is the prevalence of layer-by-layer bonding techniques, which result in limitations relating to the applicable materials and geometries and limited repeatability. In this work, we present an improved approach, using three dimensional (3D) laser lithography for the direct integration of a functional part-the membrane-into a closed-channel system. We show that it allows the freely choice of the geometry of the membrane and its integration into a complete organ-on-a-chip system. Considerations relating to sample preparation, the writing process, and the final preparation for operation are given. Overall, we consider that the broader application of 3D laser lithography in organ-on-a-chip fabrication is the next logical step in this field's evolution.

Femtosecond laser based 3D nanolithography is a powerful tool for fabricating various functional micro- and nano-objects. In this work we present several advances needed to push it from the laboratory level use to the industrial production lines. First, linear stage and galvo-scanners synchronization is employed to produce stitch-free mm-sized structures. Furthermore, it is shown that by varying objective numerical apertures (NA) from 1.4 NA to 0.45 NA, voxel size can be tuned in the range from sub μm to tens of mm, resulting in structuring rates between 1809 μm 3 /s and 313312 μm 3 /s at 1 cm/s translation velocity achieved via simultaneous movement of linear stages and scanners. Discovered voxel/throughput scaling peculiarities show good agreement to ones acquired with numerical modeling. Furthermore, support-free 3D printing of complex structures is demonstrated. It is achieved by choosing pre-polymer that is in hard gel form during laser writing and acts as a dissolvable support during manufacturing. All of this is combined to fabricate micromechanical structures. First, 1:40 aspect ratio cantilever and 1.5 mm diameter single-helix spring capable of sustaining extreme deformations for prolonged movement times (up to 10000 deformation cycles) are shown. Then, free-movable highly articulated intertwined micromechanical spider and squids (overall size up to 10 mm) are printed and their movement is tested. The presented results are discussed in the broader sense, touching on the stitching/throughput dilemma and comparing it to the standard microstereolithography. It is shown where multiphoton polymerization can outpace standard stereolithography in terms of throughput while still maintaining superior resolution and higher degree of freedom in terms of printable geometries.