Generating human skeletal myoblast spheroids for vascular myogenic tissue engineering.

Engineered myogenic microtissues derived from human skeletal myoblasts offer unique opportunities for varying skeletal muscle tissue engineering applications, such asin vitrodrug-testing and disease modelling. However, more complex models require the incorporation of vascular structures, which remains to be challenging. In this study, myogenic spheroids were generated using a high-throughput, non-adhesive micropatterned surface. Since monoculture spheroids containing human skeletal myoblasts were unable to remain their integrity, co-culture spheroids combining human skeletal myoblasts and human adipose-derived stem cells were created. When using the optimal ratio, uniform and viable spheroids with enhanced myogenic properties were achieved. Applying a pre-vascularization strategy, through addition of endothelial cells, resulted in the formation of spheroids containing capillary-like networks, lumina and collagen in the extracellular matrix, whilst retaining myogenicity. Moreover, sprouting of endothelial cells from the spheroids when encapsulated in fibrin was allowed. The possibility of spheroids, from different maturation stages, to assemble into a more large construct was proven by doublet fusion experiments. The relevance of using three-dimensional microtissues with tissue-specific microarchitecture and increased complexity, together with the high-throughput generation approach, makes the generated spheroids a suitable tool forin vitrodrug-testing and human disease modeling.

Tuning the Phenotype of Cartilage Tissue Mimics by Varying Spheroid Maturation and Methacrylamide-Modified Gelatin Hydrogel Characteristics.

In hybrid bioprinting of cartilage tissue constructs, spheroids are used as cellular building blocks and combined with biomaterials for dispensing. However, biomaterial intrinsic cues can deeply affect cell fate and to date, the influence of hydrogel encapsulation on spheroid viability and phenotype has received limited attention. This study assesses this need and unravels 1) how the phenotype of spheroid-laden constructs can be tuned through adjusting the hydrogel physico-chemical properties and 2) if the spheroid maturation stage prior to encapsulation is a determining factor for the construct phenotype. Articular chondrocyte spheroids with a cartilage specific extracellular matrix (ECM) are generated and different maturation stages, early-, mid-, and late-stage (3, 7, and 14 days, respectively), are harvested and encapsulated in 10, 15, or 20 w/v% methacrylamide-modified gelatin (gelMA) for 14 days. The encapsulation of immature spheroids do not lead to a cartilage-like ECM production but when more mature mid- or late-stage spheroids are combined with a certain concentration of gelMA, a fibrocartilage-like as well as a hyaline cartilage-like phenotype can be induced. As a proof of concept, late-stage spheroids are bioprinted using a 10 w/v% gelMA-Irgacure 2959 solution with the aim to test the processing potential of the spheroid-laden bioink.