Development of a device useful to reproducibly produce large quantities of viable and uniform stem cell spheroids with controlled diameters.

Three-dimensional cellular aggregates can mimic the natural microenvironment of tissues and organs and obtaining them through controlled and reproducible processes is mandatory for scaling up and implementing drug cytotoxicity and efficacy tests, as well as tissue engineering protocols. The purpose of this work was to develop and evaluate the performance of a device with two different geometries fabricated by additive manufacturing. The methodology was based on casting a microwell array insert using a non-adhesive hydrogel to obtain highly regular microcavities to standardize spheroid formation and morphology. Spheroids of dental pulp stem cells, bone marrow stromal cells and embryonic stem cells showing high cell viability and average diameters of around 253, 220, and 500 µm, respectively, were produced using the device with the geometry considered most adequate. The cell aggregates showed sphericity indexes above 0.9 and regular surfaces (solidity index higher than 0.96). Around 1000 spheroids could be produced in a standard six-well plate. Overall, these results show that this method facilitates obtaining a large number of uniform, viable spheroids with pre-specified average diameters and through a low-cost and reproducible process for a myriad of applications.

Comparison of the properties of membranes produced with alginate and chitosan from mushroom and from shrimp.

Dense and porous chitosan-alginate membranes (1:1 in mass) useful as coverages of skin wounds treated through cell therapy were produced using chitosan of different chain sizes from fungal (white mushrooms) and animal (shrimp shells) sources. Porous materials were obtained by adding the surfactant Poloxamer 188 to the formulations. The influence of chitosan type on membranes physicochemical properties and toxicity to fibroblasts was evaluated. Porosity was noticed to be more pronounced in membranes obtained with fungal chitosan and increased with its molecular mass. These formulations showed the highest values of thickness, roughness, opacity, liquid uptake and water vapor permeability. The membranes were not toxic to fibroblasts, but the lowest cytotoxicity values (0.16-0.21%) were observed for membranes prepared with fungal chitosan in the presence of surfactant. In conclusion, it is possible to replace chitosan from animal sources by chitosan of fungal origin to produce membranes with negligible cytotoxicity while maintaining appropriate physicochemical properties.