Accelerated and Improved Differentiation of Retinal Organoids from Mouse Pluripotent Stem Cells in Rotating-Wall Bioreactors. Mus musculus

Pluripotent stem cells can be differentiated into three-dimensional (3D) retinal organoids, with major cell types self-patterning into a polarized, laminated architecture. In static cultures, organoid development may be hindered by limitations in diffusion of oxygen and nutrients. Herein, we report a bioprocess using rotating-wall bioreactors (RWB) to culture retinal organoids derived from mouse pluripotent stem cells. Organoids in RWB demonstrate enhanced proliferation, with well-defined morphology and improved differentiation of neurons including ganglion cells and S-cone photoreceptors. Furthermore, RWB organoids at day (D)25 reveal similar maturation and transcriptome profile as those at D32 in static culture, closely recapitulating spatiotemporal development of postnatal day 6 mouse retina in vivo. Interestingly, however, retinal organoids do not differentiate further under any in vitro condition tested here, suggesting additional requirements for functional maturation. Our studies demonstrate that bioreactors can accelerate and improve organoid growth and differentiation for modeling retinal disease and evaluation of therapies. Overall design: RNA-seq characterization of embryonic stem cell derived retina organoids cultured from intact static suspension culture (SSCi) from day(D) 18 through D32, dissected static suspension culture (SSCd) from D18 through D28, and from rotating-wall bioreactor (RWB) from D18 through D25. Experiments were performed either twice or three times and considered biological replicates. Total RNA was purified from tissue homogenates using TriPure isolation reagent (Roche, Basel, Switzerland). RNA quantity was assessed with a NanoDrop 1000 spectrophotometer (Thermo Scientific, Rockville, MD). Libraries were constructed using the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego CA) and paired-end sequenced at 125 bases on a HiSeq 2500.