Highly viscous bioinks offer great advantages for the three-dimensional fabrication of cell-laden constructs by microextrusion printing. However, no standardised method of mixing a high viscosity biomaterial ink and a cell suspension has been established so far, leading to non-reproducible printing results. A novel method for the homogeneous and reproducible mixing of the two components using a mixing unit connecting two syringes is developed and investigated. Several static mixing units, based on established mixing designs, were adapted and their functionality was determined by analysing specific features of the resulting bioink. As a model system, we selected a highly viscous ink consisting of fresh frozen human blood plasma, alginate, and methylcellulose, and a cell suspension containing immortalized human mesenchymal stem cells. This bioink is crosslinked after fabrication. A pre-crosslinked gellan gum-based bioink providing a different extrusion behaviour was introduced to validate the conclusions drawn from the model system. For characterisation, bioink from different zones within the mixing device was analysed by measurement of its viscosity, shape fidelity after printing and visual homogeneity. When taking all three parameters into account, a comprehensive and reliable comparison of the mixing quality was possible. In comparison to the established method of manual mixing inside a beaker using a spatula, a significantly higher proportion of viable cells was detected directly after mixing and plotting for both bioinks when the mixing unit was used. A screw-like mixing unit, termed “HighVisc”, was found to result in a homogenous bioink after a low number of mixing cycles while achieving high cell viability rates.

The world population is growing and alternative ways of satisfying the increasing demand for meat are being explored, such as using animal cells for the fabrication of cultured meat. Edible biomaterials are required as supporting structures. Hence, we chose agarose, gellan and a xanthan-locust bean gum blend (XLB) as support materials with pea and soy protein additives and analyzed them regarding material properties and biocompatibility. We successfully built stable hydrogels containing up to 1% pea or soy protein. Higher amounts of protein resulted in poor handling properties and unstable gels. The gelation temperature range for agarose and gellan blends is between 23–30 °C, but for XLB blends it is above 55 °C. A change in viscosity and a decrease in the swelling behavior was observed in the polysaccharide-protein gels compared to the pure polysaccharide gels. None of the leachates of the investigated materials had cytotoxic effects on the myoblast cell line C2C12. All polysaccharide-protein blends evaluated turned out as potential candidates for cultured meat. For cell-laden gels, the gellan blends were the most suitable in terms of processing and uniform distribution of cells, followed by agarose blends, whereas no stable cell-laden gels could be formed with XLB blends.

Adipose tissue is related to the development and manifestation of multiple diseases, demonstrating the importance of suitable in vitro models for research purposes. In this study, adipose tissue lobuli were explanted, cultured, and used as an adipose tissue control to evaluate in vitro generated adipose tissue models. During culture, lobule exhibited a stable weight, lactate dehydrogenase, and glycerol release over 15 days. For building up in vitro adipose tissue models, we adapted the biomaterial gelatin methacryloyl (GelMA) composition and handling to homogeneously mix and bioprint human primary mature adipocytes (MA) and adipose-derived stem cells (ASCs), respectively. Accelerated cooling of the bioink turned out to be essential for the homogeneous distribution of lipid-filled MAs in the hydrogel. Last, we compared manual and bioprinted GelMA hydrogels with MA or ASCs and the explanted lobules to evaluate the impact of the printing process and rate the models concerning the physiological reference. The viability analyses demonstrated no significant difference between the groups due to additive manufacturing. The staining of intracellular lipids and perilipin A suggest that GelMA is well suited for ASCs and MA. Therefore, we successfully constructed physiological in vitro models by bioprinting MA-containing GelMA bioinks.

Due to its wide-ranging endocrine functions, adipose tissue influences the whole body’s metabolism. Engineering long-term stable and functional human adipose tissue is still challenging due to the limited availability of suitable biomaterials and adequate cell maturation. We used gellan gum (GG) to create manual and bioprinted adipose tissue models because of its similarities to the native extracellular matrix and its easily tunable properties. Gellan gum itself was neither toxic nor monocyte activating. The resulting hydrogels exhibited suitable viscoelastic properties for soft tissues and were stable for 98 days in vitro. Encapsulated human primary adipose-derived stem cells (ASCs) were adipogenically differentiated for 14 days and matured for an additional 84 days. Live-dead staining showed that encapsulated cells stayed viable until day 98, while intracellular lipid staining showed an increase over time and a differentiation rate of 76% between days 28 and 56. After 4 weeks of culture, adipocytes had a univacuolar morphology, expressed perilipin A, and secreted up to 73% more leptin. After bioprinting establishment, we demonstrated that the cells in printed hydrogels had high cell viability and exhibited an adipogenic phenotype and function. In summary, GG-based adipose tissue models show long-term stability and allow ASCs maturation into functional, univacuolar adipocytes.