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

2021 ◽  
Author(s):  
Capuzzo Arnaud Martino ◽  
Daniele Vigo
Keyword(s):  
Fluorescence Intensity ◽  
Regulatory Networks ◽  
3D Structure ◽  
3D Models ◽  
Live Imaging ◽  
Live Cells ◽  
Straight Line Passing ◽  
3D Cultures ◽  
Research Activities

ABSTRACTOne 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.

scholarly journals Microfluidic Live-Imaging Technology to Perform Research Activities in 3D Models

2021 ◽  
Author(s):  
Arnaud Martino Capuzzo ◽  
Daniele Vigo
Keyword(s):  
Fluorescence Intensity ◽  
Regulatory Networks ◽  
3D Structure ◽  
Live Imaging ◽  
Two Dimensions ◽  
Live Cells ◽  
Straight Line Passing ◽  
3D Cultures ◽  
Research Activities

Morphological dissimilarity and its evolution over time are one of the most unexpected variations found when comparing cell cultures in 2D and 3D. Monolayer cells appear to flatten in the lower part of the plate, adhering to and spreading in the horizontal plane while not extending vertically. Consequently, cells developed in two dimensions have a forced apex-basal polarity. Co-cultivation and crosstalking between multiple cell types, which control development and formation in the in vivo counterpart, are possible in 3D cultures. With or without a scaffold matrix, 3D model culture may exhibit more in vivo-like morphology and physiology. 3D cultures mimic relevant physiological cellular processes, transforming them into one-of-a-kind drug screening platforms. The structures and dynamics of regulatory networks, which are increasingly studied with live-imaging microscopy, must be considered to help and guarantee the functional maintenance of a 3D structure. However, commercially available technologies that can be used for current laboratory needs are minimal, despite the need to make it easier to acquire cellular kinetics with high spatial and temporal resolution, in order to improve visual efficiency and, as a result, experimentation performance. The CELLviewer is a newly developed multi-technology instrument that integrates and synchronizes the work of various scientific disciplines. The aim of this study is to test the device using two different models: a single Jurkat cell and an MCF-7 spheroid. The two models are loaded into the microfluidic cartridge for each experiment after they have been grown and captured in time-lapse for a total of 4 hours. The samples used are tracked under the operation of the optics after adaptive autofocus, while slipping inside the cartridge chamber, and the 3D rotation was successfully obtained experimentally. The MitoGreen dye, a fluorescence marker selectively permeable to live cells, was then used to determine cell viability. To measure the model diameter, construct fluorescence intensity graphs along a straight line passing through the cell, and visualize the spatial fluorescence intensity distribution in 3D, ImageJ software was used.

scholarly journals Microfluidic Live-Imaging Technology to Perform Research Activities in 3D Models

2021 ◽  
Author(s):  
Arnaud Martino Capuzzo ◽  
Daniele Vigo
Keyword(s):  
Fluorescence Intensity ◽  
Regulatory Networks ◽  
3D Structure ◽  
Time Lapse ◽  
3D Models ◽  
Live Cells ◽  
Straight Line Passing ◽  
3D Cultures ◽  
Research Activities

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.

scholarly journals Microfluidic Live Imaging Technology to Perform Research Activities in 3D Models

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Capuzzo Arnaud Martino
Keyword(s):  
Fluorescence Intensity ◽  
Regulatory Networks ◽  
3D Structure ◽  
Live Imaging ◽  
Two Dimensions ◽  
Live Cells ◽  
Straight Line Passing ◽  
3D Cultures ◽  
Research Activities

Morphological dissimilarity and its evolution over time are one of the most unexpected variations found when comparing cell cultures in 2D and 3D. Monolayer cells appear to flatten in the lower part of the plate, adhering to and spreading in the horizontal plane while not extending vertically. Consequently, cells developed in two dimensions have a forced apex-basal polarity. Co-cultivation and crosstalking between multiple cell types, which control development and formation in the in vivo counterpart, are possible in 3D cultures. With or without a scaffold matrix, 3D model culture may exhibit more in vivo-like morphology and physiology. 3D cultures mimic relevant physiological cellular processes, transforming them into one-of-akind drug screening platforms. The structures and dynamics of regulatory networks, which are increasingly studied with live-imaging microscopy, must be considered to help and guarantee the functional maintenance of a 3D structure. However, commercially available technologies that can be used for current laboratory needs are minimal, despite the need to make it easier to acquire cellular kinetics with high spatial and temporal resolution, in order to improve visual efficiency and, as a result, experimentation performance. The CELLviewer is a newly developed multi-technology instrument that integrates and synchronizes the work of various scientific disciplines. The aim of this study is to test the device using two different models: a single Jurkat cell and an MCF-7 1 spheroid. The two models are loaded into the microfluidic cartridge for each experiment after they have been grown and captured in time-lapse for a total of 4 hours. The samples used are tracked under the operation of the optics after adaptive autofocus, while slipping inside the cartridge chamber and the 3D rotation was successfully obtained experimentally. The MitoGreen dye, a fluorescence marker selectively permeable to live cells, was then used to determine cell viability. To measure the model diameter, construct fluorescence intensity graphs along a straight line passing through the cell, and visualize the spatial fluorescence intensity distribution in 3D, Image J software was used.

scholarly journals Visual Barcodes for Multiplexing Live Microscopy-Based Assays

2020 ◽  
Author(s):  
Tom Kaufman ◽  
Erez Nitzan ◽  
Nir Firestein ◽  
Miriam Ginzberg ◽  
Seshu Iyengar ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Single Cells ◽  
Live Imaging ◽  
Live Cells ◽  
Balance Growth ◽  
Complex Biological System ◽  
Clonal Identity

Abstract While multiplexing samples using DNA barcoding revolutionized the pace of biomedical discovery, multiplexing of live imaging-based applications has been limited by the number of fluorescent proteins that can be deconvoluted using common microscopy equipment. To address this limitation we developed visual barcodes that discriminate the clonal identity of single cells by targeting different fluorescent proteins to specific subcellular locations. We demonstrate that deconvolution of these barcodes is highly accurate and robust to many cellular perturbations. We then used visual barcodes to generate ‘Signalome’ cell-lines by multiplexing live reporters to monitor the simultaneous activity in 12 branches of signaling, in live cells, at single cell resolution, over time. Using the ‘Signalome’ we identified two distinct clusters of signaling pathways that balance growth and proliferation, emphasizing the importance of growth homeostasis as a central organizing principle in cancer signaling. The ability to multiplex samples in live imaging applications, both in vitro and in vivo may allow better high-content characterization of complex biological system

scholarly journals Pre-Clinical In Vitro Models Used in Cancer Research: Results of a Worldwide Survey

Cancers ◽  
2021 ◽  
Vol 13 (23) ◽  
pp. 6033
Author(s):  
Sarai Martinez-Pacheco ◽  
Lorraine O’Driscoll
Keyword(s):  
Cancer Research ◽  
Three Dimensional ◽  
3D Cell Culture ◽  
Cost Effective ◽  
Cell Types ◽  
3D Models ◽  
In Vitro Models ◽  
3D Cultures

To develop and subsequently get cancer researchers to use organotypic three-dimensional (3D) models that can recapitulate the complexity of human in vivo tumors in an in vitro setting, it is important to establish what in vitro model(s) researchers are currently using and the reasons why. Thus, we developed a survey on this topic, obtained ethics approval, and circulated it throughout the world. The survey was completed by 101 researchers, across all career stages, in academia, clinical or industry settings. It included 40 questions, many with multiple options. Respondents reported on their field of cancer research; type of cancers studied; use of two-dimensional (2D)/monolayer, 2.5D and/or 3D cultures; if using co-cultures, the cell types(s) they co-culture; if using 3D cultures, whether these involve culturing the cells in a particular way to generate spheroids, or if they use additional supports/scaffolds; techniques used to analyze the 2D/2.5D/3D; and their downstream applications. Most researchers (>66%) only use 2D cultures, mainly due to lack of experience and costs. Despite most cancer researchers currently not using the 3D format, >80% recognize their importance and would like to progress to using 3D models. This suggests an urgent need to standardize reliable, robust, reproducible methods for establishing cost-effective 3D cell culture models and their subsequent characterization.

Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

1996 ◽  
Vol 54 ◽  
pp. 730-731
Author(s):  
E. D. Salmon ◽  
J. C. Waters ◽  
C. Waterman-Storer
Keyword(s):  
Imaging System ◽  
Ccd Camera ◽  
Transmitted Light ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Optical Efficiency ◽  
Multiple Band ◽  
Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

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