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 liveimaging 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 2 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.

"In Vitro 3d Model of Female Reproductive Tract: An Overview and Future Aspect"

“…Intensive efforts to create various freezing/thawing procedures and cryoprotective substances finally resulted in the first human pregnancy from a frozen embryo in 1983, which unfortunately ended in premature membrane rupture and pregnancy termination at 24 weeks of gestation. (A M Capuzzo, Vigo [52]). Despite the setback, cryopreservation technology advanced in the 1980s, leading to an increase in embryo survival and conception rates (Equipment and Materials) [53].…”

From the Classification of Stem Cells to the Release of Potential in Cell Therapies: Limits, Considerations and Future Aspects in Regenerative Medicine

“…Currently few technologies are available to generate large quantities of cultured stem cells and keep them incubating. We have previously successfully tested the combination of CELLviewer [65] with Spin∞, a new bioreactor still in the prototype stage [66].…”

Initial refinement of data from video‐based single‐cell tracking

“…The advantage of using these models for tracking single cells is their low cost and reproducibility as compared to three‐dimensional (3D) platforms [ 30 , 31 , 32 ]. 2D models can easily integrate subsequent biochemical analysis and act as surrogate measurements for 3D situations [ 33 ].…”

“…3D models are under active development to better represent the complexity of living organisms during in vitro research [ 34 , 35 , 36 , 37 ]. However, they still do not recapitulate micro‐environmental factors, being only reductionist of the in vivo counterpart [ 29 , 33 ]. 3D cell culture models are currently application specific and experiments with them are difficult to check for repeatability [ 29 ].…”

“…3D cell culture models are currently application specific and experiments with them are difficult to check for repeatability [ 29 ]. Current 3D platforms do not allow acquisition of cellular kinetics with a high spatial and temporal resolution over a long period of time [ 33 , 38 ]. High‐content screening (HCS) platforms are emerging, however, visualization of 3D structures growing within complex geometrical structures remain still a big challenge mainly due to optical light scattering, light absorption, and poor light penetration with prolonged imaging acquisition times [ 29 ].…”