Ungrin, Mark D.Kondro, Douglas A.2022-11-152020-09-14Kondro, D. A. (2020). Scaling Cellular Aggregate Production in a Microwell System (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.http://hdl.handle.net/1880/115479https://dx.doi.org/10.11575/PRISM/40446Microtissues, in the form of cellular aggregates, offer a promising format for cell-based therapies, such as the treatment of type 1 diabetes with beta cell replacement. One major challenge is producing a sufficient quantity of microtissue for clinical use. For instance, type 1 diabetes requires approximately 1 million cellular aggregates for treatment of a single individual. Static culture methods of producing aggregates, such as the microwell system, offer an accessible format of producing high quality, size-controlled cellular aggregates, however they cannot easily generate the quantity required for a clinical treatment. This thesis scales an existing microwell format by producing a robust, self-contained, multilayered microwell bioreactor capable of generating over 250,000 cellular aggregates in a well plate footprint. The design of the microwell bioreactor consists of a sealed chamber where cells aggregate on thin sheets of microwells in the device. The microwells are made with an oxygen permeable polymer to facilitate gas exchange in the sealed system, that was demonstrated to deliver approximately 4 times the oxygen to cells in comparison with the well plate format. The design process utilizes hot-embossing, allowing for efficient and scalable manufacturing, with a demonstrated cycle time as low as 15 seconds per microwell sheet. The final device is robust, capable of withstanding centrifugation up to 2000 g, and autoclaving sterilization. Further, the protocol for cell culture is simplified as it allows for removal of >97% of media during a media change without disturbing aggregates, and removal of >98% of cellular aggregates after long term culturing. Biological validation and demonstration of clinical utility was performed using 12 donor islet batches. The microwell bioreactor could scale the production of functional pseudoislets in the form of size-controlled cellular aggregates. Pseudoislets were demonstrated to have indistinguishable functional performance in compared to previously published methods. Applicability of this novel system was also demonstrated for other diseases by successfully differentiating pluripotent stem cells and aggregating testicular organoids. The unique design of the microwell bioreactor provides a straightforward tool to enable researchers to immediately scale static culture experiments, allowing for translation of cell-based therapies to clinical use.enUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.Tissue EngineeringType 1 diabetesMicromanufacturingGas diffusionBiology--CellEngineering--BiomedicalEngineering--Mechanicaldoctoral thesis