Establishment of 3D culture protocols for the maintenance and expansion of human pluripotent stem cell aggregates in a low scale platform and in the DASbox® Mini-Bioreactor System
KAUST DepartmentBiological and Environmental Science and Engineering (BESE) Division
Embargo End Date2023-07-27
Permanent link to this recordhttp://hdl.handle.net/10754/679909
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Access RestrictionsAt the time of archiving, the student author of this thesis opted to temporarily restrict access to it. The full text of this thesis will become available to the public after the expiration of the embargo on 2023-07-27.
AbstractThe human Embryonic Stem Cells (hESCs) and human induced Pluripotent Stem Cells (hiPSCs) have offered numerous advantages including but not limited to model diseases, high-throughput drug screening, and regenerative purposes. However, the employment of monolayer cultures has not been sufficient to mimic the in vivo stem cells niche. Thus, three-dimensional suspension cultures have helped us to advance our knowledge and ease the development of the human organs’ counterparts, commonly referred as organoids. Currently, the challenge is the generation of homogenous and reproducible human Pluripotent Stem Cell (hPSC) aggregates, the basic cellular unit to derive organoids. To date, the Ultra-Low Attachment (ULA) 6-well plates have been routinary used for the hPSC aggregates formation, which mainly relies on the inhibition of the Rho-associated kinase (ROCK) pathway resulting in the enhancement of cell survival coming from cryopreserved stocks or when passaging. However, little is known in this regard when analyzing the aggregate formation of hPSCs with two widely used compounds: RevitaCellTM Supplement and Y27632. Importantly, due to the high demand required from the regenerative medicine, I aimed to upscale the hPSC aggregates production in the DASbox® Mini-Bioreactor System. In this thesis, I established protocols for the hPSC aggregates formation by using two different types of media in two platforms being the ULA 6-well plates and the DASbox® Mini-Bioreactor System. In addition, I demonstrated that monolayer confluence cultures before single cell inoculations are paramount for the formation of bona fide hPSC aggregates in healthy and X aneuploid hiPSCs, precisely two hESCs and five hiPSCs.
CitationHernandez-Bautista, C. A. (2022). Establishment of 3D culture protocols for the maintenance and expansion of human pluripotent stem cell aggregates in a low scale platform and in the DASbox® Mini-Bioreactor System [KAUST Research Repository]. https://doi.org/10.25781/KAUST-0898S