In multicellular organisms, cells are organized in a 3-dimensional framework and this is essential for organogenesis and tissue morphogenesis. Systems to recapitulate 3D cell growth are therefore vital for understanding development and cancer biology. Cells organized in 3D environments can evolve certain phenotypic traits valuable to physiologically relevant models that cannot be accessed in 2D culture. Cellular spheroids constitute an important aspect of in vitro tumor biology and they are usually prepared using the hanging drop method. Here a 3D printed approach is demonstrated to fabricate bespoke hanging drop devices for the culture of tumor cells. The design attributes of the hanging drop device take into account the need for high-throughput, high efficacy in spheroid formation, and automation. Specifically, in this study, custom-fit, modularized hanging drop devices comprising of inserts (Q-serts) were designed and fabricated using fused filament deposition (FFD). The utility of the Q-serts in the engineering of unicellular and multicellular spheroids-synthetic tumor microenvironment mimics (STEMs)—was established using human (cancer) cells. The culture of spheroids was automated using a pipetting robot and bioprinted using a custom bioink based on carboxylated agarose to simulate a tumor microenvironment (TME). The spheroids were characterized using light microscopy and histology. They showed good morphological and structural integrity and had high viability throughout the entire workflow. The systems and workflow presented here represent a user-focused 3D printing-driven spheroid culture platform which can be reliably reproduced in any research environment and scaled to- and on-demand. The standardization of spheroid preparation, handling, and culture should eliminate user-dependent variables, and have a positive impact on translational research to enable direct comparison of scientific findings.

Due to the limited regenerative capacity of injured cartilage, surgical intervention using engineered cellular constructs or autologous cell implantation is the best accredited approach to prevent further degeneration and promote a regenerative microenvironment. Advancements in additive manufacturing present opportunities for graft customization through enhanced scaffold design. In bioprinting, an additive manufacturing process, the "bioink" serves as the medium to carry cells but also as a scaffold by imparting form and mechanical attributes to the printed object. In this study, the impact of cross-linked gelatin microparticles (GMPs) on rheological properties and printability of carboxylated agarose (CA) bioink as well as matrix deposition by human nasal chondrocytes (hNCs) was investigated. The introduction of GMPs yielded stiffer bioink formulations, with lower sol-gel transitions that retained the exceptional printability of CA. GMPs served as foci for the attachment of hNCs, improving cellular distribution and bridging the deposited extracellular matrix. After 4 weeks in chondrogenic culture, GMPs containing printed constructs showed enhanced toughness approaching that of the lower end of the spectrum of native cartilage tissue. The incorporation of proteinaceous microparticles might serve as a general concept to promote cellular function in polysaccharide-based bioinks and opens another avenue for engineering 3D-bioprinted microenvironments.

Microfluidic devices (µFDs) have been explored extensively in drug screening and studying cellular processes such as migration and metastasis. However, the fabrication and implementation of microfluidic devices pose cost and logistical challenges that limit wider-spread adoption. Despite these challenges, light-based 3D printing offers a potential alternative to device fabrication. This study reports on the development of millifluidic devices (MiFDs) for disease modeling and elucidates the methods and implications of the design, production, and testing of 3D-printed MiFDs. It further details how such millifluidic devices can be cost-efficiently and effortlessly produced. The MiFD was developed through an iterative process with analytical tests (flow tests, leak tests, cytotoxicity assays, and microscopic analyses), driving design evolution and determination of the suitability of the devices for disease modeling and cancer research. The design evolution also considered flow within tissues and replicates interstitial flow between the main flow path and the modules designed to house and support organ-mimicking cancer cell spheroids. Although the primary stereolithographic (SLA) resin used in this study showed cytotoxic potential despite its biocompatibility certifications, the MiFDs possessed essential attributes for cell culturing. In summary, SLA 3D printing enables the production of MiFDs as a cost-effective, rapid prototyping alternative to standard µFD fabrication for investigating disease-related processes.

Over the past decade, three-dimensional bioprinting (3DBP) has evolved into a versatile processing tool for engineering tissues. The key component, the bioink, can be composed of many different hydrogel-forming polymers, which are mostly performant in either mechanical or biological properties but seldom both. Carboxylated agarose (CA) was combined here with collagen type 1 to simultaneously satisfy both properties and combine their attributes, without compromising on either; the result is an innovative, hybrid bioink. It can be printed with high accuracy, good layer-to-layer adhesion, and rapid gelation, enabling overhang printing. Scanning electron microscopy (SEM) and fluorescently labeled collagen demonstrated that the two components mixed well, resulting in uniformly distributed collagen fibrils and a double network. From the biological perspective, a bioink must exhibit cell-adhesion moieties to maintain proliferation and metabolism, which we could ensure through the collagen component. Here, we present an example strategy for combining an inert polysaccharide with bioactive collagen, two polymers with opposing gelation conditions, which yields a bioink that possesses beneficial properties from both without compromising the features of either. The interpenetrating structure of both molecules synergistically balances the mechanical strength of CA and the biological functionality of collagen.