title: 3D high throughput screening and profiling of embryoid bodies in thermoformed microwell plates.
abstract: 3D organoids using stem cells to study development and disease are now widespread. These models are powerful to mimic in vivo situations but are currently associated with high variability and low throughput. For biomedical research, platforms are thus necessary to increase reproducibility and allow high-throughput screens (HTS). Here, we introduce a microwell platform, integrated in standard culture plates, for functional HTS. Using micro-thermoforming, we form round-bottom microwell arrays from optically clear cyclic olefin polymer films, and assemble them with bottom-less 96-well plates. We show that embryonic stem cells aggregate faster and more reproducibly (centricity, circularity) as compared to a state-of-the-art microwell array. We then run a screen of a chemical library to direct differentiation into primitive endoderm (PrE) and, using on-chip high content imaging (HCI), we identify molecules, including regulators of the cAMP pathway, regulating tissue size, morphology and PrE gene activity. We propose that this platform will benefit to the systematic study of organogenesis in vitro.
authors.affiliation: Merln Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht, The Netherlands. erikvrij@gmail.com nicolasrivron@gmail.com.
authors.forename: E J
authors.initials: EJ
authors.lastname: Vrij
authors.forename: S
authors.initials: S
authors.lastname: Espinoza
authors.forename: M
authors.initials: M
authors.lastname: Heilig
authors.forename: A
authors.initials: A
authors.lastname: Kolew
authors.forename: M
authors.initials: M
authors.lastname: Schneider
authors.forename: C A
authors.initials: CA
authors.lastname: van Blitterswijk
authors.forename: R K
authors.initials: RK
authors.lastname: Truckenmüller
authors.forename: N C
authors.initials: NC
authors.lastname: Rivron
mesh_terms: D000818:Animals; D002449:Cell Aggregation / Q000187:drug effects; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D058732:Embryoid Bodies / Q000166:cytology* / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D057166:High-Throughput Screening Assays / Q000379:methods*; D007700:Kinetics; D051379:Mice; D055616:Microtechnology / Q000379:methods*; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D020796:Receptor, Platelet-Derived Growth Factor alpha / Q000378:metabolism; D015203:Reproducibility of Results; D013696:Temperature
chemical_list: D047428:Protein Kinase Inhibitors; D000242:Cyclic AMP; D020796:Receptor, Platelet-Derived Growth Factor alpha; D017868:Cyclic AMP-Dependent Protein Kinases