Dynamic full-field optical coherence tomography module adapted to commercial microscopes allows longitudinal in vitro cell culture study.

Dynamic full-field optical coherence tomography (D-FFOCT) has recently emerged as a label-free imaging tool, capable of resolving cell types and organelles within 3D live samples, whilst monitoring their activity at tens of milliseconds resolution. Here, a D-FFOCT module design is presented which can be coupled to a commercial microscope with a stage top incubator, allowing non-invasive label-free longitudinal imaging over periods of minutes to weeks on the same sample. Long term volumetric imaging on human induced pluripotent stem cell-derived retinal organoids is demonstrated, highlighting tissue and cell organization processes such as rosette formation and mitosis as well as cell shape and motility. Imaging on retinal explants highlights single 3D cone and rod structures. An optimal workflow for data acquisition, postprocessing and saving is demonstrated, resulting in a time gain factor of 10 compared to prior state of the art. Finally, a method to increase D-FFOCT signal-to-noise ratio is demonstrated, allowing rapid organoid screening.

Bankable human iPSC-derived retinal progenitors represent a valuable source of multipotent cells.

Retinal progenitor cells (RPCs) are the source of all retinal cell types during retinogenesis. Until now, the isolation and expansion of RPCs has been at the expense of their multipotency. Here, we report simple methods and media for the generation, expansion, and cryopreservation of human induced pluripotent stem-cell derived-RPCs (hiRPCs). Thawed and passed hiRPCs maintained biochemical and transcriptional RPC phenotypes and their ability to differentiate into all retinal cell types. Specific conditions allowed the generation of large cultures of photoreceptor precursors enriched up to 90% within a few weeks and without a purification step. Combined RNA-seq analysis between hiRPCs and retinal organoids identified genes involved in developmental or degenerative retinal diseases. Thus, hiRPC lines could provide a valuable source of retinal cells for cell-based therapies or drug discovery and could be an advanced cellular tool to better understand retinal dystrophies.

Dynamic full-field optical coherence tomography allows live imaging of retinal pigment epithelium stress model.

Retinal degenerative diseases lead to the blindness of millions of people around the world. In case of age-related macular degeneration (AMD), the atrophy of retinal pigment epithelium (RPE) precedes neural dystrophy. But as crucial as understanding both healthy and pathological RPE cell physiology is for those diseases, no current technique allows subcellular in vivo or in vitro live observation of this critical cell layer. To fill this gap, we propose dynamic full-field OCT (D-FFOCT) as a candidate for live observation of in vitro RPE phenotype. In this way, we monitored primary porcine and human stem cell-derived RPE cells in stress model conditions by performing scratch assays. In this study, we quantified wound healing parameters on the stressed RPE, and observed different cell phenotypes, displayed by the D-FFOCT signal. In order to decipher the subcellular contributions to these dynamic profiles, we performed immunohistochemistry to identify which organelles generate the signal and found mitochondria to be the main contributor to D-FFOCT contrast. Altogether, D-FFOCT appears to be an innovative method to follow degenerative disease evolution and could be an appreciated method in the future for live patient diagnostics and to direct treatment choice.