Quantitative pigment extraction analysis for human pluripotent stem cell derived retinal pigment epithelial cells

Abstract

Pigmentation is a typical feature for retinal pigment epithelial cells (RPE), and each RPE cell represents individual characteristics. Although, type and intensity of pigmentation may vary, still most cells are pigmented to some extent. In this study, we setup a standardized quantitative melanin content analysis for human embryonic stem cell (hESC) derived RPEs (hESC-RPE) and compared this method to an image based pigment quantification technique. The optical quantification of pigmentation was done from micrographs of hESC-RPE. The extracted pigment was quantitated with spectral analysis, with UV-Vis spectroscopy, and the size-distribution with dynamic light scattering. The results revealed that the measured particle sizes of extracted melanin corresponded to known sizes extracted melanosomes. In addition, the optical pigmentation and absolute melanin concentration were clearly correlating. Our data suggests that image analysis and quantitative melanin content analysis can be interchangeably utilized in pigmentation quantitation: if the relative pigmentation of hESC-RPE cells is desired to be estimated with a non-invasive method then image analysis is the choice of method, but if the exact amount of melanin needs to be evaluated, then the new melanin extraction method presented here, should be chosen.

Deep learning predicts function of live retinal pigment epithelium from quantitative microscopy

“…Thus, the methodology developed here starts with a rigorous, reproducible absorbance imaging method using a bright-field microscope. Absorption imaging has been reported in the literature (26)(27)(28), but the QBAM technology developed here can be implemented on any standard bright-field microscope, and it uses real-time, automated, statistically robust methods to provide high confidence in image quality and reproducibility. The advantage to using absorbance rather than raw pixel intensities is that absorbance is an absolute measure of light attenuation (29).…”