A High‐Throughput Automated Confocal Microscopy Platform for Quantitative Phenotyping of Nanoparticle Uptake and Transport in Spheroids

Abstract

There is a high demand for advanced, image‐based, automated high‐content screening (HCS) approaches to facilitate phenotypic screening in 3D cell culture models. A major challenge lies in retaining the resolution of fine cellular detail but at the same time imaging multicellular structures at a large scale. In this study, a confocal microscopy‐based HCS platform in optical multiwell plates that enables the quantitative morphological profiling of populations of nonuniform spheroids obtained from HT‐29 human colorectal cancer cells is described. This platform is then utilized to demonstrate a quantitative dissection of the penetration of synthetic nanoparticles (NP) in multicellular 3D spheroids at multiple levels of scale. A pilot RNA interference‐based screening validates this methodology and identifies a subset of RAB GTPases that regulate NP trafficking in these spheroids. This technology is suitable for high‐content phenotyping in 3D cell‐based screening, providing a framework for nanomedicine drug development as applied to translational oncology.

Transport of nanoprobes in multicellular spheroids

“…There are numerous studies on the penetration of specific nanoparticles through spheroids. 29,30 In comparison with these reports, we present a systematic study on the rheological characteristics of the ECM in spheroids at different length scales. Table 2.…”

Robotics, microfluidics, nanotechnology and AI in the synthesis and evaluation of liposomes and polymeric drug delivery systems

“…In particular, automated confocal microscopes containing incubators allow live-cell imaging and characterisation of fluorescent components [ 68 ]. Despite the high accuracy and precision, manual confocal fluorescence microscopy is a time-consuming task when analysing hundreds of samples [ 69 ]. The need for automated microscopy has increased when the possibility of measuring a 384-well-plate in a single field-of-view in only several minutes became a reality [ 70 ].…”

High-throughput screening of nanoparticles in drug delivery

“… 98,122,123 In the last 30 years, some studies have used three-dimensional (3D) cell cultures in order to recapitulate the complexity of the in vivo organization. These 3D cell cultures can adopt different formats such as multicellular structures derived from more than one type of cell (e.g., micro-tissues and co-culture systems), cell aggregates from a single cell type (also termed spheroids), 124 and organoids that are derived from cells differentiated from a progenitor cell population with capacity to form organ-like structures. 125 Many of HTS studies documented so far with nanomaterial libraries have not explored the use of 3D cell cultures or intact organs ex vivo .…”