Three-dimensional (3D) bioprinting is one of the most promising methodologies that are currently in development for the replacement of animal experiments. Bioprinting and most alternative technologies rely on animal-derived materials, which compromises the intent of animal welfare and results in the generation of chimeric systems of limited value. The current study therefore presents the first bioprinted liver model that is entirely void of animal-derived constituents. Initially, HuH-7 cells underwent adaptation to a chemically defined medium (CDM). The adapted cells exhibited high survival rates (85–92%) after cryopreservation in chemically defined freezing media, comparable to those preserved in standard medium (86–92%). Xeno-free bioink for 3D bioprinting yielded liver models with high relative cell viability (97–101%), akin to a Matrigel-based liver model (83–102%) after 15 days of culture. The established xeno-free model was used for toxicity testing of a marine biotoxin, okadaic acid (OA). In 2D culture, OA toxicity was virtually identical for cells cultured under standard conditions and in CDM. In the xeno-free bioprinted liver model, 3-fold higher concentrations of OA than in the respective monolayer culture were needed to induce cytotoxicity. In conclusion, this study describes for the first time the development of a xeno-free 3D bioprinted liver model and its applicability for research purposes.

Background: The natural extracellular matrix (ECM) provides the optimal environment for cells. Many enzymatic or non-enzymatic based strategies to extract ECM proteins from tissues were published over the last years. However, every single isolation strategy reported so far is associated with specific bottlenecks. Experiment: In this study, frequently used strategies to isolate extracellular matrix (ECM) from human placenta or adipose tissue using Tris-, serum, or pepsin-based buffers were compared. The resulting ECM proteins were biochemically characterized by analysis of cellular remnants using HOECHST DNA staining, glycosaminoglycan (GAG) content by dimethylemethylene blue (DMMB), visualization of protein bands using SDS PAGE analysis combined with amino acid quantification and assessment of the pro-angiogenic profile using an angiogenesis array. Results: Tris-NaCl extracted ECM proteins showed a high heterogenic degree of extracted proteins, bioactive growth factors and GAGS, but no collagen-I. Active serum extracted ECM showed significant lower DNA remnants when compared to the Tris-NaCl isolation strategy. Pepsin-extracted ECM was rich in collagen-I and low amounts of remaining bioactive growth factors. This strategy was most effective to reduce DNA amounts when compared to the other isolation strategies. Pepsin-extracted ECM from both tissues easily gelled at 37°C, whereas the other extracted ECM strategies did not gel at 37°C (Tris-NaCl: liquid; serum: sponge). Conclusions: All relevant characteristics (DNA residues, ECM diversity and bioactivity, shape) of the extracted ECM proteins highly depend on its isolation strategy and could still be optimized.

Background: There is critical unmet need for new vascularized tissues to support or replace injured tissues and organs. Various synthetic and natural materials were already established for use of 2D and 3D in vitro neovascularization assays, however, they still cannot mimic the complex functions of the sum of the extracellular matrix (ECM) in native, intact tissue. Currently, this issue is only addressed by artificial products like MatrigelTM, which comprises of a complex mixture of ECM proteins, extracted from animal tumor tissue. Despite its outstanding bioactivity, the isolation from tumor tissue hinders its translation into clinical applications. Since non-human ECM proteins may cause immune reactions, as are frequently observed in clinical trials, human ECM proteins represent the best option when aiming for clinical applications, Experiment: Here, we describe an effective method of isolating a human placenta substrate (hpS) that induces the spontaneous formation of an interconnected network of green fluorescence labeled human umbilical vein endothelial cells (gfpHUVEC) in vitro. The substrate was biochemically characterized by using a combination of bicinchoninic acid assay (BCA), DNA and glycosaminoglycan (GAG) content assays, sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis and Western blot, angiogenesis arrays, chromatographic thrombin detection, HPLC-based amino acid quantification analysis and assessment of antimicrobial properties. Results: 2D in vitro cell culture experiments have been performed to determine the vasculogenic potential of hpS, which demonstrated that cell networks developed on hpS show a significantly higher degree of complexity (number of tubules/junctions; total/mean tube length) when compared to MatrigelTM. As 3D cell culture techniques represent a more accurate representation of the in vivo condition, the substrate was 3D solidified using various natural polymers. 3D in vitro vasculogenesis assays have been performed by seeding gfpHUVEC in a hpS-fibrinogen clot. Conclusions: Concluding, hpS provides a potent human-material-based alternative to xenogenic-material-based biomaterials for vascularization strategies in tissue engineering.