Microfluidic Live-Imaging technology to perform research activities in 3D models

Abstract

One of the most surprising differences observed when comparing cell cultures in 2D and 3D is morphological dissimilarity and their evolution over time. Cells grown in a monolayer tend to flatten in the lower part of the plate adhering to and spreading in the horizontal plane without expanding in the vertical dimension. The result is that cells grown in 2D have a forced apex-basal polarity. 3D cultures support co-cultivation and crosstalking between multiple cell types, which regulate development and formation in the in vivo counterpart. 3D models culture, with or without a scaffold matrix, can exhibit more in vivo-like morphology and physiology. 3D cultures recapitulate relevant physiological cellular processes, transforming into unique platforms for drug screening. To support and guarantee the functional maintenance of a 3D structure, one must consider the structures and dynamics of regulatory networks, increasingly studied with live-imaging microscopy. However, commercially available technologies that can be used for current laboratory needs are limited, although there is a need to facilitate the acquisition of cellular kinetics with a high spatial and temporal resolution, to elevate visual performance and consequently that of experimentation. The CELLviewer is a newly conceived and developed multi-technology instrumentation, combining and synchronizing the work of different scientific disciplines. This work aims to test the system with two models: the first model is a single Jurkat cell while the second is an MCF-7 spheroid. After having grown both models, the two models used are loaded into the microfluidic cartridge for each experiment and recorded in time-lapse for a total of 4 hours. After adaptive autofocus, when sliding inside the cartridge chamber, the samples used are tracked under the action of the optics and the 3D rotation was experimentally successfully obtained. A cell viability assessment was then used using the MitoGreen dye, a fluorescence marker selectively permeable to live cells. The ImageJ software was used to: calculate the model diameter, create fluorescence intensity graphs along a straight line passing through the cell, visualize the spatial fluorescence intensity distribution in 3D.