Speaker
Description
"Introduction: For a viable and compliant clinical translation of tissue-engineered products, the adoption of automated technologies has been acknowledged as a prerequisite. Recently, the use of chondrogenic microtissue and organoid assemblies has shown promising results in long-bone defect regeneration through endochondral ossification[1]. Hence, automated biomanufacturing technologies able to culture and handle these tissue building blocks are of great interest. Here, we present an automation strategy through the use of different robotics for (i) media change during differentiation, (ii) plate movement, and (iii) image-based picking of microtissues for enabling spheroid-specific quality control.
Methods: Periosteum derived cells were seeded in a commercial microwell platform (AggrewellTM800, 1000 cells/spheroid or AggrewellTM400, 250 cells/spheroid). They were cultured for 21 days in chondrogenic medium. Media change of microtissues in microwell platforms requires controlled pipetting to avoid microtissue displacement and suspension leading to uncontrolled fusion. We set up a design of experiment (DoE) for aspiration and dispension speed during automated media changes. Then, we investigated the effect of robotic plate handling and automated imaging on spheroid movement for different size spheroids. After 21 days, we created ectopic implants through a controlled fusion of 900 large, or 3600 small spheroids to assess the bone-forming capacity, which is evaluated through µCT and safranin-O histochemistry. Subsequently, the automatic cell-screening and -picking system CellCelector™ was used to select and transfer single spheroids in a controlled manner. Here, spheroids were automatically selected based on the presence of only 1 spheroid/well and/or size via image-based analysis.
Results: The first DoE revealed that dispension speed, rather than aspiration speed, had a significant effect on local spheroid movement during media changes. The second DoE showed that smaller microtissues were more susceptible to movement than larger spheroids. Also, plate handling had a significant impact on overall movement. After 21 days, spheroids from all conditions were able to mineralize ectopically. Finally, using the CellCelectorTM, we were able to pick and place single spheroids without affecting their viability.
Discussion: Bottom-up engineering of skeletal implants requires a vast amount of diffusion-unlimited spheroids as building blocks. Culturing these for multiple weeks to achieve the desired differentiation is a complex process that requires expert personnel to avoid spheroid movement that leads to uncontrolled fusion. To enable scale-up and increase process robustness, we demonstrate the development of an integrated bioprocess for culturing and manipulation of cartilaginous spheroids. We anticipate the progressive substitution of manual operations with automated solutions for the manufacturing of microtissue-based living implants.
Reference: [1] G. Nilsson Hall, L.F. Mendes, C. Gklava, L. Geris, F.P. Luyten, I. Papantoniou, Developmentally Engineered Callus Organoid Bioassemblies Exhibit Predictive In Vivo Long Bone Healing, Adv. Sci. 7 (2020) 1–16. doi:10.1002/advs.201902295."
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