Development of a robotic-assisted handling and manipulation system for the high-scale bioproduction of 3D-bioprinted organ-on-a-chip devices.

Organs-on-a-chip (OoCs) have proven to mimic the basic physiological behavior of organs and the influence of therapeutics on them in greater detail than conventional models, resulting in enormous projected market growth rates. However, the breakthrough to profitable commercialization of that technology has not yet been achieved, partly because the production process chain is characterized by a high proportion of manual laboratory work. The present work addresses this point. Utilizing affordable components, a demonstrator was developed that can be integrated into an existing 3D-bioprinting system and enables the automated production of perfusion-ready OoC devices starting from pre-fabricated injection-molded microfluidic chips. To this end, a corresponding process chain was first defined, and an expandable, configurable algorithm was developed and validated in the form of a finite state machine (FSM). This algorithm controls a modified 4-axis robot arm that covers the steps upstream and downstream of the printing process in the manufacturing process and achieves success rates of up to 100 %. A virtual interface between the robot and printer enables mutual communication and full integration of the algorithm into the process chain. Steps that pose a challenge for the automation of the process chain and appropriate countermeasures and optimizations were identified. This lays the foundation for scaling and standardizing the automated production of OoCs.

High-Scale 3D-Bioprinting Platform for the Automated Production of Vascularized Organs-on-a-Chip.

3D bioprinting possesses the potential to revolutionize contemporary methodologies for fabricating tissue models employed in pharmaceutical research and experimental investigations. This is enhanced by combining bioprinting with advanced organs-on-a-chip (OOCs), which includes a complex arrangement of multiple cell types representing organ-specific cells, connective tissue, and vasculature. However, both OOCs and bioprinting so far demand a high degree of manual intervention, thereby impeding efficiency and inhibiting scalability to meet technological requirements. Through the combination of drop-on-demand bioprinting with robotic handling of microfluidic chips, a print procedure is achieved that is proficient in managing three distinct tissue models on a chip within only a minute, as well as capable of consecutively processing numerous OOCs without manual intervention. This process rests upon the development of a post-printing sealable microfluidic chip, that is compatible with different types of 3D-bioprinters and easily connected to a perfusion system. The capabilities of the automized bioprint process are showcased through the creation of a multicellular and vascularized liver carcinoma model on the chip. The process achieves full vascularization and stable microvascular network formation over 14 days of culture time, with pronounced spheroidal cell growth and albumin secretion of HepG2 serving as a representative cell model.

Influence of the physico-chemical bioink composition on the printability and cell biological properties in 3D-bioprinting of a liver tumor cell line.

The selection of a suitable matrix material is crucial for the development of functional, biomimetic tissue and organ models. When these tissue models are fabricated with 3D-bioprinting technology, the requirements do not only include the biological functionality and physico-chemical properties, but also the printability. In our work, we therefore present a detailed study of seven different bioinks with the focus on a functional liver carcinoma model. Agarose, gelatin, collagen and their blends were selected as materials based on their benefits for 3D cell culture and Drop-on-Demand (DoD) bioprinting. The formulations were characterized for their mechanical (G' of 10-350 Pa) and rheological (viscosity 2-200 Pa*s) properties as well as albumin diffusivity (8-50 µm2/s). The cellular behavior was exemplarily shown for HepG2 cells by monitoring viability, proliferation and morphology over 14 days, while the printability on a microvalve DoD printer was evaluated by drop volume monitoring in flight (100-250 nl), camera imaging of the wetting behavior and microscopy of the effective drop diameter (700 µm and more). We did not observe negative effects on cell viability or proliferation, which is due to the very low shear stresses inside the nozzle (200-500 Pa). With our method, we could identify the strengths and weaknesses of each material, resulting in a material portfolio. By specifically selecting certain materials or blends, cell migration and possible interaction with other cells can be directed as indicated by the results of our cellular experiments.

Fabrication of biomimetic networks using viscous fingering in flexographic printing.

Mammalian tissue comprises a plethora of hierarchically organized channel networks that serve as routes for the exchange of liquids, nutrients, bio-chemical cues or electrical signals, such as blood vessels, nerve fibers, or lymphatic conduits. Despite differences in function and size, the networks exhibit a similar, highly branched morphology with dendritic extensions. Mimicking such hierarchical networks represents a milestone in the biofabrication of tissues and organs. Work to date has focused primarily on the replication of the vasculature. Despite initial progress, reproducing such structures across scales and increasing biofabrication efficiency remain a challenge. In this work, we present a new biofabrication method that takes advantage of the viscous fingering phenomenon. Using flexographic printing, highly branched, inter-connective channel structures with stochastic, biomimetic distribution and dendritic extensions can be fabricated with unprecedented efficiency. Using gelatin (5%-35%) as resolvable sacrificial material, the feasability of the proposed method is demonstrated on the example of a vascular network. By selectively adjusting the printing velocity (0.2-1.5 m s-1), the anilox roller dip volume (4.5-24 ml m-2) as well as the shear viscosity of the printing material used (10-900 mPas), the width of the structures produced (30-400 µm) as well as their distance (200-600 µm) can be specifically determined. In addition to the flexible morphology, the high scalability (2500-25 000 mm2) and speed (1.5 m s-1) of the biofabrication process represents an important unique selling point. Printing parameters and hydrogel formulations are investigated and tuned towards a process window for controlled fabrication of channels that mimic the morphology of small blood vessels and capillaries. Subsequently, the resolvable structures were casted in a hydrogel matrix enabling bulk environments with integrated channels. The perfusability of the branched, inter-connective structures was successfully demonstrated. The fabricated networks hold great potential to enable nutrient supply in thick vascularized tissues or perfused organ-on-a-chip systems. In the future, the concept can be further optimized and expanded towards large-scale and cost-efficient biofabrication of vascular, lymphatic or neural networks for tissue engineering and regenerative medicine.

Investigation and comparison of resin materials in transparent DLP-printing for application in cell culture and organs-on-a-chip.

Organs-on-a-Chip (OOCs) have recently led to major discoveries and a better understanding of 3D cell organization, cell-cell interactions and tissue response to drugs and biological cues. However, their complexity and variability are still limited by the available fabrication technology. Transparent, cytocompatible and high-resolution 3D-printing could overcome these limitations, offering a flexible and low-cost alternative to soft lithography. Many advances have been made in stereolithography printing regarding resin formulation and the general printing process, but a systematic analysis of the printing process steps, employed resins and post-treatment procedures with a strong focus on the requirements in OOCs is missing. To fill this gap, this work provides an in-depth analysis of three different resin systems in comparison to polystyrene (PS) and poly(dimethylsiloxane) (PDMS), which can be considered the gold-standards in cell culture and microfluidics. The resins were characterized with respect to transparency, cytocompatibility and print resolution. These properties are not only governed by the resin composition, but additionally by the post-treatment procedure. The investigation of the mechanical (elastic modulus ∼2.2 GPa) and wetting properties (∼60° native / 20° plasma treated) showed a behavior very similar to PS. In addition, the absorbance of small molecules was two orders of magnitude lower in the applied resins (diffusion constant ∼0.01 µm2 s-1) than for PDMS (2.5 µm2 s-1), demonstrating the intrinsic suitability of these materials for OOCs. Raman spectroscopy and UV/VIS spectrophotometry revealed that post-treatment increased monomer conversion up to 2 times and removed photo initiator residues, leading to an increased transparency of up to 50% and up to 10-times higher cell viability. High magnification fluorescence imaging of HUVECs and L929 cells cultivated on printed dishes shows the high optical qualities of prints fabricated by the Digital Light Processing (DLP) printer. Finally, components of microfluidic chips such as high-aspect ratio pillars and holes with a diameter of 50 µm were printed. Concluding, the suitability of DLP-printing for OOCs was demonstrated by filling a printed chip with a cell-hydrogel mixture using a microvalve bioprinter, followed by the successful cultivation under perfusion. Our results highlight that DLP-printing has matured into a robust fabrication technology ready for application in extensive and versatile OOC research.

Biosynthetic, biomimetic, and self-assembled vascularized Organ-on-a-Chip systems.

Organ-on-a-Chip (OOC) devices have seen major advances in the last years with respect to biological complexity, physiological composition and biomedical relevance. In this context, integration of vasculature has proven to be a crucial element for long-term culture of thick tissue samples as well as for realistic pharmacokinetic, toxicity and metabolic modelling. With the emergence of digital production technologies and the reinvention of existing tools, a multitude of design approaches for guided angio- and vasculogenesis is available today. The underlying production methods can be categorized into biosynthetic, biomimetic and self-assembled vasculature formation. The diversity and importance of production approaches, vascularization strategies as well as biomaterials and cell sourcing are illustrated in this work. A comprehensive technological review with a strong focus on the challenge of producing physiologically relevant vascular structures is given. Finally, the remaining obstacles and opportunities in the development of vascularized Organ-on-a-Chip platforms for advancing drug development and predictive disease modelling are noted.

Nanomechanical sub-surface mapping of living biological cells by force microscopy.

Atomic force microscopy allows for the nanomechanical surface characterization of a multitude of types of materials with highest spatial precision in various relevant environments. In recent years, researchers have refined this methodology to analyze living biological materials in vitro. The atomic force microscope thus has become an essential instrument for the (in many cases) non-destructive, high-resolution imaging of cells and visualization of their dynamic mechanical processes. Mapping force versus distance curves and the local evaluation of soft samples allow the operator to "see" beneath the sample surface and to capture the local mechanical properties. In this work, we combine atomic force microscopy with fluorescence microscopy to investigate cancerous epithelial breast cells in culture medium. With unprecedented spatial resolution, we provide tomographic images for the local elasticity of confluent layers of cells. For these particular samples, a layer of higher elastic modulus located directly beneath the cell membrane in comparison with the average elastic properties was observed. Strikingly, this layer appears to be perforated at unique locations of the sample surface of weakest mechanical properties where distinct features were visible permitting the tip to indent farthest into the cell's volume. We interpret this layer as the cell membrane mechanically supported by the components of the cytoskeleton that is populated with sites of integral membrane proteins. These proteins act as breaking points for the indenter thus explaining the mechanical weakness at these locations. In contrast, the highest mechanical strength of the cell was found at locations of the cell cores as cross-checked by fluorescence microscopy images of staining experiments, in particular at nucleoli sites as the cumulative elastic modulus there comprises cytoskeletal features and the tight packing ribosomal DNA of the cell.