RESUMEN
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.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Cuerpos Embrioides/citología , Cuerpos Embrioides/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Microtecnología/métodos , Temperatura , Animales , Agregación Celular/efectos de los fármacos , Línea Celular , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Cinética , Ratones , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Reproducibilidad de los ResultadosRESUMEN
A promising approach to bone tissue engineering lies in the use of perfusion bioreactors where cells are seeded and cultured on scaffolds under conditions of enhanced nutrient supply and removal of metabolic products. Fluid flow alterations can stimulate cell activity, making the engineering of tissue more efficient. Most bioreactor systems are used to culture cells on thin scaffold discs. In clinical use, however, bone substitutes of large dimensions are needed. In this study, MG63 osteoblast-like cells were seeded on large porous PLA/glass scaffolds with a custom developed perfusion bioreactor system. Cells were seeded by oscillating perfusion of cell suspension through the scaffolds. Applicable perfusion parameters for successful cell seeding were determined by varying fluid flow velocity and perfusion cycle number. After perfusion, cell seeding, the cell distribution, and cell seeding efficiency were determined. A fluid flow velocity of 5 mm/s had to be exceeded to achieve a uniform cell distribution throughout the scaffold interior. Cell seeding efficiencies of up to 50% were achieved. Results suggested that perfusion cycle number influenced cell seeding efficiency rather than fluid flow velocities. The cell seeding conducted is a promising basis for further long term cell culture studies in large porous scaffolds.