The effect of continuous long-term illumination with visible light in different spectral ranges on mammalian cells.

One of the biggest challenges in tissue engineering and regenerative medicine is to ensure oxygen supply of cells in the (temporary) absence of vasculature. With the vision to exploit photosynthetic oxygen production by microalgae, co-cultivated in close vicinity to oxygen-consuming mammalian cells, we are searching for culture conditions that are compatible for both sides. Herein, we investigated the impact of long-term illumination on mammalian cells which is essential to enable photosynthesis by microalgae: four different cell types-primary human fibroblasts, dental pulp stem cells, and osteoblasts as well as the murine beta-cell line INS-1-were continuously exposed to warm white light, red or blue light over seven days. We observed that illumination with red light has no adverse effects on viability, metabolic activity and growth of the cells whereas exposure to white light has deleterious effects that can be attributed to its blue light portion. Quantification of intracellular glutathione did not reveal a clear correlation of this effect with an enhanced production of reactive oxygen species. Finally, our data indicate that the cytotoxic effect of short-wavelength light is predominantly a direct effect of cell illumination; photo-induced changes in the cell culture media play only a minor role.

Assessing non-synthetic crosslinkers in biomaterial inks based on polymers of marine origin to increase the shape fidelity in 3D extrusion printing.

In the past decade, there has been significant progress in 3D printing research for tissue engineering (TE) using biomaterial inks made from natural and synthetic compounds. These constructs can aid in the regeneration process after tissue loss or injury, but achieving high shape fidelity is a challenge as it affects the construct's physical and biological performance with cells. In parallel with the growth of 3D bioprinting approaches, some marine-origin polymers have been studied due to their biocompatibility, biodegradability, low immunogenicity, and similarities to human extracellular matrix components, making them an excellent alternative to land mammal-origin polymers with reduced disease transmission risk and ethical concerns. In this research, collagen from shark skin, chitosan from squid pens, and fucoidan from brown algae were effectively blended for the manufacturing of an adequate biomaterial ink to achieve a printable, reproducible material with a high shape fidelity and reticulated using four different approaches (phosphate-buffered saline, cell culture medium, 6% CaCl2, and 5 mM Genipin). Materials characterization was composed by filament collapse, fusion behavior, swelling behavior, and rheological and compressive tests, which demonstrated favorable shape fidelity resulting in a stable structure without deformations, and interesting shear recovery properties around the 80% mark. Additionally, live/dead assays were conducted in order to assess the cell viability of an immortalized human mesenchymal stem cell line, seeded directly on the 3D printed constructs, which showed over 90% viable cells. Overall, the Roswell Park Memorial Institute cell culture medium promoted the adequate crosslinking of this biopolymer blend to serve the TE approach, taking advantage of its capacity to hamper pH decrease coming from the acidic biomaterial ink. While the crosslinking occurs, the pH can be easily monitored by the presence of the indicator phenol red in the cell culture medium, which reduces costs and time.

Selection of a suitable photosynthetically active microalgae strain for the co-cultivation with mammalian cells.

Preventing hypoxic zones in 3D bioprinted mammalian cell-laden constructs using an internal oxygen supply could enable a more successful cultivation both in vitro and in vivo. In this study, the suitability of green microalgae as photosynthetic oxygen generators within bioprinted constructs was evaluated by defining and investigating important parameters for a successful co-culture. First, we assessed the impact of light-necessary for photosynthesis-on two non-light adapted mammalian cell types and defined red-light illumination and a temperature of 37°C as essential factors in a co-culture. The four thermotolerant microalgae strains Chlorella sorokiniana, Coelastrella oocystiformis, Coelastrella striolata, and Scenedesmus sp. were cultured both in suspension culture and 3D bioprinted constructs to assess viability and photosynthetic activity under these defined co-culture conditions. Scenedesmus sp. proved to be performing best under red light and 37°C as well as immobilized in a bioprinted hydrogel based on alginate. Moreover, the presence of the antibiotic ampicillin and the organic carbon-source glucose, both required for mammalian cell cultures, had no impact on bioprinted Scenedesmus sp. cultures regarding growth, viability, and photosynthetic activity. This study is the first to investigate the influence of mammalian cell requirements on the metabolism and photosynthetic ability of different microalgal strains. In a co-culture, the strain Scenedesmus sp. could provide a stable oxygenation that ensures the functionality of the mammalian cells.

Think outside the box: 3D bioprinting concepts for biotechnological applications - recent developments and future perspectives.

3D bioprinting - the fabrication of geometrically complex 3D structures from biocompatible materials containing living cells using additive manufacturing technologies - is a rapidly developing research field with a broad range of potential applications in fundamental research, regenerative medicine and industry. Currently, research into 3D bioprinting is mostly focused on new therapeutic concepts for the treatment of injured or degenerative tissue by fabrication of functional tissue equivalents or disease models, utilizing mammalian cells. However, 3D bioprinting also has an enormous potential in biotechnology. Due to the defined spatial arrangement of biologically active (non-mammalian) cells in a biomaterial matrix, reaction compartments can be designed according to specific needs, or co-cultures of different cell types can be realized in a highly organized manner to exploit cell-cell interactions. Thus, 3D bioprinting technology can enable new biotechnological concepts, for example, by implementing perfusion systems while protecting shear sensitive cells or performing cascaded bioreactions. Here, we review the use of 3D bioprinting to manufacture defined 3D microenvironments for biotechnological applications using bacteria, fungi, microalgae, plant cells and co-cultures of different cell types. We discuss recent approaches to apply 3D bioprinting in biotechnological applications and - as it is a particular challenge - concepts for the real-time monitoring of the physiological state, growth and metabolic activity of the embedded cells in 3D bioprinted constructs. With these insights, we outline new applications of 3D bioprinting in biotechnology, engineered living materials and space research.

Homogeneous and Reproducible Mixing of Highly Viscous Biomaterial Inks and Cell Suspensions to Create Bioinks.

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.