Cartilage microtissues are promising tissue modules for bottom up biofabrication of implants leading to bone defect regeneration. Hitherto, most of the protocols for the development of these cartilaginous microtissues have been carried out in static setups, however, for achieving higher scales, dynamic process needs to be investigated. In the present study, we explored the impact of suspension culture on the cartilage microtissues in a novel stirred microbioreactor system. To study the effect of the process shear stress, experiments with three different impeller velocities were carried out. Moreover, we used mathematical modeling to estimate the magnitude of shear stress on the individual microtissues during dynamic culture. Identification of appropriate mixing intensity allowed dynamic bioreactor culture of the microtissues for up to 14 days maintaining microtissue suspension. Dynamic culture did not affect microtissue viability, although lower proliferation was observed as opposed to the statically cultured ones. However, when assessing cell differentiation, gene expression values showed significant upregulation of both Indian Hedgehog (IHH) and collagen type X (COLX), well known markers of chondrogenic hypertrophy, for the dynamically cultured microtissues. Exometabolomics analysis revealed similarly distinct metabolic profiles between static and dynamic conditions. Dynamic cultured microtissues showed a higher glycolytic profile compared with the statically cultured

The field of tissue engineering aspires to provide clinically relevant solutions for patients through the integration of developmental engineering principles with a bottom-up manufacturing approach. However, the manufacturing of cell-based advanced therapy medicinal products is hampered by protocol complexity, lack of non-invasive critical quality controls, and dependency on animal-derived components for tissue differentiation. We investigate a serum-free, chemically defined, xeno- and lipid-free chondrogenic differentiation medium to generate bone-forming callus organoids. Our results show an increase in microtissue homogeneity during prolonged differentiation and the high quality of in vivo bone-forming organoids. The low protein content of the culture medium potentially allows for the monitoring of relevant secreted biomarkers as (critical) quality attributes. Together, we envisage that this xeno- and lipid-free chondrogenic medium is compatible with industrial scale-up and automation while facilitating the implementation of non-invasive imaging and the use of quality control parameters based on secreted biomarkers.

Automated technologies are attractive for enhancing a robust manufacturing of tissue engineered products for clinical translation. In this work, we present an automation strategy using a robotics platform for media changes of cartilaginous microtissues cultured in static microwell platforms. We use an automated image analysis pipeline to extract microtissue displacements and morphological features, which serve as input for statistical factor analysis. To minimize microtissue displacement and suspension leading to uncontrolled fusion, we performed a mixed factorial DoE on liquid handling parameters for large and small microwell platforms. As a result, 144 images, with 51 471 spheroids could be processed automatically. The automated imaging workflow takes 2 minutes per image, and it can be implemented for on-line monitoring of microtissues, thus allowing informed decision making during manufacturing. We found that time in culture is the main factor for microtissue displacements, explaining 10 % of the displacements. Aspiration and dispension speed were not significant at manual speeds or beyond, with an effect size of 1 %. We defined optimal needle placement and depth for automated media changes and we suggest that robotic plate handling could improve the yield and homogeneity in size of microtissue cultures. After three weeks culture, increased expression of COL2A1 confirmed chondrogenic differentiation and RUNX2 shows no osteogenic specification. Histological analysis showed the secretion of cartilaginous extracellular matrix. Furthermore, microtissue-based implants were capable of forming mineralized tissues and bone after four weeks of ectopic implantation in nude mice. We demonstrate the development of an integrated bioprocess for culturing and manipulation of cartilaginous microtissues. We anticipate the progressive substitution of manual operations with automated solutions for manufacturing of microtissue-based living implants.

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