Bioinks for Space Missions: The Influence of Long-Term Storage of Alginate-Methylcellulose-Based Bioinks on Printability as well as Cell Viability and Function.

Bioprinting is considered a key technology for future space missions and is currently being established on the International Space Station (ISS). With the aim to perform bioink production as a critical and resource-consuming preparatory step already on Earth and transport a bioink cartridge "ready to use" to the ISS, the storability of bioinks is investigated. Hydrogel blends based on alginate and methylcellulose are laden with either green microalgae of the species Chlorella vulgaris or with different human cell lines including immortilized human mesenchymal stem cells, SaOS-2 and HepG2, as well as with primary human dental pulp stem cells. The bioinks are filled into printing cartridges and stored at 4°C for up to four weeks. Printability of the bioinks is maintained after storage. Viability and function of the cells embedded in constructs bioprinted from the stored bioinks are investigated during subsequent cultivation: The microalgae survive the storage period very well and show no loss of growth and functionality, however a significant decrease is visible for human cells, varying between the different cell types. The study demonstrates that storage of bioinks is in principle possible and is a promising starting point for future research, making complex printing processes more effective and reproducible.

Viability and Functionality of Neonatal Porcine Islet-like Cell Clusters Bioprinted in Alginate-Based Bioinks.

The transplantation of pancreatic islets can prevent severe long-term complications in diabetes mellitus type 1 patients. With respect to a shortage of donor organs, the transplantation of xenogeneic islets is highly attractive. To avoid rejection, islets can be encapsulated in immuno-protective hydrogel-macrocapsules, whereby 3D bioprinted structures with macropores allow for a high surface-to-volume ratio and reduced diffusion distances. In the present study, we applied 3D bioprinting to encapsulate the potentially clinically applicable neonatal porcine islet-like cell clusters (NICC) in alginate-methylcellulose. The material was additionally supplemented with bovine serum albumin or the human blood plasma derivatives platelet lysate and fresh frozen plasma. NICC were analysed for viability, proliferation, the presence of hormones, and the release of insulin in reaction to glucose stimulation. Bioprinted NICC are homogeneously distributed, remain morphologically intact, and show a comparable viability and proliferation to control NICC. The number of insulin-positive cells is comparable between the groups and over time. The amount of insulin release increases over time and is released in response to glucose stimulation over 4 weeks. In summary, we show the successful bioprinting of NICC and could demonstrate functionality over the long-term in vitro. Supplementation resulted in a trend for higher viability, but no additional benefit on functionality was observed.

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.

The Secretome of Hypoxia Conditioned hMSC Loaded in a Central Depot Induces Chemotaxis and Angiogenesis in a Biomimetic Mineralized Collagen Bone Replacement Material.

The development of biomaterials with intrinsic potential to stimulate endogenous tissue regeneration at the site of injury is a main demand on future implants in regenerative medicine. For critical-sized bone defects, an in situ tissue engineering concept is devised based on biomimetic mineralized collagen scaffolds. These scaffolds are functionalized with a central depot loaded with a signaling factor cocktail, obtained from secretome of hypoxia-conditioned human mesenchymal stem cells (MSC). Therefore, hypoxia-conditioned medium (HCM)-production is standardized and adapted to achieve high signaling factor-yields; a concentration protocol based on dialysis and freeze-drying is established to enable the integration of sufficient and defined amounts into the depot. In humid milieu-as after implantation-signaling factors are released by forming a chemotactic gradient, inducing a directed migration of human bone marrow stroma cells (hBMSC) into the scaffold. Angiogenic potential, determined by coculturing human umbilical vein endothelial cells (HUVEC) with osteogenically induced hBMSC shows prevascular structures, which sprout throughout the interconnected pores in a HCM-concentration-dependent manner. Retarded release by alginate-based (1 vol%) depots, significantly improves sprouting-depth and morphology of tubular structures. With the intrinsic potential to supply attracted cells with oxygen and nutrients, this bioactive material system has great potential for clinical translation.

3D Bioprinting of Functional Islets of Langerhans in an Alginate/Methylcellulose Hydrogel Blend.

Transplantation of pancreatic islets is a promising strategy to alleviate the unstable blood-glucose control that some patients with diabetes type 1 exhibit and has seen many advances over the years. Protection of transplanted islets from the immune system can be accomplished by encapsulation within a hydrogel, the most investigated of which is alginate. In this study, islet encapsulation is combined with 3D extrusion bioprinting, an additive manufacturing method which enables the fabrication of 3D structures with a precise geometry to produce macroporous hydrogel constructs with embedded islets. Using a plottable hydrogel blend consisting of clinically approved ultrapure alginate and methylcellulose (Alg/MC) enables encapsulating pancreatic islets in macroporous 3D hydrogel constructs of defined geometry while retaining their viability, morphology, and functionality. Diffusion of glucose and insulin in the Alg/MC hydrogel is comparable to diffusion in plain alginate; the embedded islets continuously produce insulin and glucagon throughout the observation and still react to glucose stimulation albeit to a lesser degree than control islets.

Investigating the effect of sterilisation methods on the physical properties and cytocompatibility of methyl cellulose used in combination with alginate for 3D-bioplotting of chondrocytes.

For both the incorporation of cells and future therapeutic applications the sterility of a biomaterial must be ensured. However, common sterilisation techniques are intense and often negatively impact on material physicochemical attributes, which can affect its suitability for tissue engineering and 3D printing. In the present study four sterilisation methods, autoclave, supercritical CO2 (scCO2) treatment, UV- and gamma (γ) irradiation were evaluated regarding their impact on material properties and cellular responses. The investigations were performed on methyl cellulose (MC) as a component of an alginate/methyl cellulose (alg/MC) bioink, used for bioprinting embedded bovine primary chondrocytes (BPCs). In contrast to the autoclave, scCO2 and UV-treatments, the γ-irradiated MC resulted in a strong reduction in alg/MC viscosity and stability after extrusion which made this method unsuitable for precise bioprinting. Gel permeation chromatography analysis revealed a significant reduction in MC molecular mass only after γ-irradiation, which influenced MC chain mobility in the Ca2+-crosslinked alginate network as well as gel composition and microstructure. With regard to cell survival and proteoglycan matrix production, the results determined UV-irradiation and autoclaving as the best candidates for sterilisation. The scCO2-treatment of MC resulted in an unfavourable cell response indicating that this method needs careful optimisation prior to application for cell encapsulation. As proven by consistent FT-IR spectra, chemical alterations could be excluded as a cause for the differences seen between MC treatments on alg/MC behaviour. This investigation provides knowledge for the development of a clinically appropriate 3D-printing-based fabrication process to produce bioengineered tissue for cartilage regeneration.

Coacervation-Mediated Combinatorial Synthesis of Biomatrices for Stem Cell Culture and Directed Differentiation.

Combinatorial screening represents a promising strategy to discover biomaterials for tailored cell culture applications. Although libraries incorporating different biochemical cues have been investigated, few simultaneously recapitulate relevant biochemical, physical, and dynamic features of the extracellular matrix (ECM). Here, a noncovalent system based on liquid-liquid phase separation (coacervation) and gelation mediated by glycosaminoglycan (GAG)-peptide interactions is reported. Multiple biomaterial libraries are generated using combinations of sulfated glycosaminoglycans and poly(ethylene glycol)-conjugated peptides. Screening these biomaterials reveals preferred biomatrices for the attachment of six cell types, including primary mesenchymal stromal cells (MSCs) and primary neural precursor cells (NPCs). Incorporation of GAGs sustains the expansion of all tested cell types comparable to standard cell culture surfaces, while osteogenic differentiation of MSC and neuronal differentiation of NPC are promoted on chondroitin and heparan biomatrices, respectively. The presented noncovalent system provides a powerful tool for developing tissue-specific ECM mimics.

Chemical structure and biological activity of a highly branched (1 → 3,1 → 6)-ß-D-glucan from Isochrysis galbana.

A highly branched (1 → 3,1 → 6)-ß-D-glucan was isolated from the microalga Isochrysis galbana Parke (Isochrysidales, Haptophyta). The polysaccharide structure was analyzed by methylation and Smith degradation, as well as by ESI and MALDI TOF mass spectrometry and NMR spectroscopy. The glucan was shown to contain a (1 → 6)-linked backbone, where every residue is substituted at position 3 by Glc, which in turn may be substituted at C-6 by a single Glc or by rather short (up to tetrasaccharide) oligosaccharide chains. All the 3-linked Glc residues are present in these side chains. In the biological activity experiments it was demonstrated that the polysaccharide directly inhibits the proliferation of U937 human leukemic monocyte lymphoma cells and therefore has potential anti-tumor activity.