Application of 3D Cell Explorer to the Analysis of Cell Cultures

Cells on Chips

Two‐dimensional (“2-D”) cell culture systems do not accurately recapitulate the structure, function, physiology of living tissues, as well as highly complex and dynamic three-dimensional (“3-D”) environments in vivo. The cell‐on‐a‐chip technology can provide micro‐scale complex structures and well‐controlled parameters to mimic the in vivo environment of cells. The 3D Cell Explorer offers the great potential of non‐invasive, 3‐D real‐time imaging of cells directly on these glass chips.

Nanopillars Glass Chip
Nanopillars Glass Chip

3D Cell Explorer images of fibroblast reticular cell seeded on a glass nanopillar array. The adhesion of the cell to the substrate is guided by the nanopillar structures. Components are digitally stained based on their specific values of their Refractive Index: cell cytoplasm in purple, nucleoli in yellow, nano-pillars in green.

3‐D Cell Culture

Hydrogels are becoming more and more popular as platforms for three‐dimensional (“3‐D”) cell culturing. 3‐D hydrogel matrices have been used for a variety of applications, including tissue engineering of micro‐organ systems, drug delivery, cytotoxicity testing, and drug screening. Moreover, 3‐D cell cultures are applied for investigating cellular physiology, stem cell differentiation, tumor models and for studying interaction mechanisms between cells and the extracellular matrix.

Engineered 3‐D extracellular matrices (such as gels and hydrogels) have been recently confirmed to have a very significant role in cell reprogramming, becoming a main actor in the generation of iPSC (Induced Pluripotent Stem Cell) as published in Nature by Matthias Lutolf’s lab at Ecole Polytechnique Fédérale de Lausanne.

Immunofluorescence combined with confocal microscopy is one on the most common ways for the study of cells embedded in 3‐D gel matrices. Nevertheless, the gel matrix can reduce the accessibility to chemicals, affecting the efficiency of permeability and increasing the needed amount of antibody and incubation time; the interaction between the 3‐D matrix and antibodies could also result in mislabeling, producing a non-specific signal.

Nanolive’s 3D Cell Explorer surpass these limitations allowing for fast and reliable imaging of cells embedded in alginate spheres with no chemical staining!

3‐D Cell Culture: Cells Embedded in Alginate Beads

HeLa cells encapsulated in alginate beads suspended in DMEM solution and visualized through a glass coverslip. The alginate beads were generated using sciDROP PICO technology mounted on a sciFLEXARRAYER S3 (SCIENION AG, Germany).

Visualizations of Microgel Beads in Multiple Modes of Illumination
Visualizations of Microgel Beads in Multiple Modes of Illumination

Microgel beads: refractive index map of Z‐stack, digitally‐stained 3‐D visualization, phase‐contrast image, fluorescence image

Arginate Bead with Cells
Arginate Bead with Cells

Arginate bead with cells: refractive index map of Z‐stack, 3‐D visualization showing digitally‐stained background, bead, cell membrane and cell nucleus

Co‐culture

A co‐culture is a cell cultivation set-up in which two or more different populations of cells are grown with some degree of contact between them. Co‐culture systems have long been used to study the interactions between cell populations and are fundamental in cell‐cell interaction studies of any kind. The 3D Cell Explorer allows for the first time a real‐time and non‐invasive long‐term co‐culture monitoring. The microscope detects the Refractive Index (“RI”) of each cell, allowing an automatic segmentation of cell populations. Moreover, Nanolive’s technology gives the possibility to study the cell interactions in 3‐D, allowing for X‐Y‐Z measurements with unprecedented nanometer resolution (resolution in X and Y is 200 nm; in Z, it is 400 nm).

B16 Mouse Melanoma Cancer Cells and Dictyostelium Amoeba Co-culture

Mouse skin melanoma cancer cells (B16, p35) were incubated overnight with Dictyostelium amoebae cells (WT1, p14), in order to visualize their interactions. Our digital staining panel allows the discrimination of mammalian cells (blue) and amoeba cells (green) based on their specific refractive index range. The RI difference (ΔRI=0.015) allows for an automatic segmentation of cell populations. We observed a simultaneous, massive mammalian cell necrosis, the reason for which remains unknown.

3D Cell Explorer Zoom-in Capabilities and X‐Y‐Z Measurements
3D Cell Explorer Zoom-in Capabilities and X‐Y‐Z Measurements of Amoeba Details

Multi-layers

On this page you can explore multi-layered cell culture as we see it: in 3‐D and stain‐free.

3D Cell Explorer allows for:

  • Real‐time, label‐free monitoring of cellular multi‐layer construction;
  • Estimation of multi‐layer thickness, homogeneity and distribution;
  • Differential staining, based on specific refractive index (“RI”), of cellular substructures in multi‐layer.
Refractive Index Map of Z‐stack, and 3‐D Visualization of Digitally‐stained Vesicles and Cellular Membrane
Refractive Index Map of Z‐stack, and 3‐D Visualization of Digitally‐stained Vesicles and Cellular Membrane

Contact inhibition is a regulatory mechanism that functions to keep fibroblast‐like cells growing in a monolayer. If a cell has plenty of available substrate space, it moves freely and replicates rapidly. This process continues until the cells occupy the entire available substratum. After that, normal cells will stop migrating and replicating (100% confluent culture). Cancer cells, in contrast, are generally insensitive to such contact inhibition. They continue moving after contact with their neighbors, migrating over adjacent cells and generating multi‐layered cultures.

Drug development requires simple in vitro models that resemble the in vivo situation. Use of such models allows reduction of experimentation on animals. These multilayered cultures could be used as relatively simple three‐dimensional systems with which to study the effects of microenvironmental conditions on anticancer drug activity.

Fibroblastic Reticular Cells Multi‐layers

Mouse Fibroblastic Reticular Cells (“FRC’s”) were grown over 100% confluency in complete DMEM medium (Dulbecco’s Modified Eagle Medium) in 35mm glass bottom culture dishes (FluoroDishes™ WPI, #FD35-100).

Low Confluency

On this page you can explore cell culture with low confluency as we see it: in 3‐D and stain‐free.

3D Cell Explorer allows for real‐time, label‐free monitoring of single cell seeding and growth.

Refractive Index Map of Z‐stack, and 3‐D Visualization of Digitally‐stained Vesicles, Cellular Membrane, Nuclear Membrane, Nuclei, and Nucleoli
Refractive Index Map of Z‐stack, and 3‐D Visualization of Digitally‐stained Vesicles, Cellular Membrane, Nuclear Membrane, Nuclei, and Nucleoli

In cell culture biology, confluence is the term commonly used as an estimate of the number of adherent cells in a culture dish or a flask, referring to the proportion of the surface which is covered by cells. For example, 50% confluency means that roughly half of the surface is covered. A regular and precise monitoring of the cell confluence status is fundamental for well‐defined experimental planning: the efficacy of many transfection protocols and subculturing procedures are strictly dependent on the cell culture confluent status.

The 3D Cell Explorer allows you to acquire 3D images of your cell cultures in real-time and in a non‐invasive way.

Lung Cancer Cells: Low Confluency

Mouse skin melanoma cancer cells (B16, p35) were grown to 25% confluency in complete DMEM medium (Dulbecco’s Modified Eagle Medium) in 35mm glass‐bottom culture dishes (FluoroDishes™ WPI, #FD35-100).

High Confluency

On this page you can explore cell culture with high confluency as we see it: in 3‐D and stain‐free.

3D Cell Explorer allows for visualization of cellular multi‐layer construction and for contact inhibition studies.

Refractive Index Map of Z‐stack, and 3‐D Visualization of Digitally‐stained Cells
Refractive Index Map of Z‐stack, and 3‐D Visualization of Digitally‐stained Cells

In cell culture biology, confluence is the term commonly used as an estimate of the number of adherent cells in a culture dish or a flask, referring to the proportion of the surface which is covered by cells. For example, 100% confluency means that the surface is completely covered by the cells, and no more room is left for the cells to grow as a monolayer. Many cell lines exhibit differences in growth rate or gene expression depending on the degree of confluence. Cells are typically passaged before becoming fully confluent in order to maintain their proliferative phenotype. Some cell types, not limited by contact inhibition, such as immortalized cells, may continue to divide and form layers on top of the parent cells. To achieve optimal and consistent results, experiments are usually performed using cells at a particular confluence, depending on the cell type.

The 3D Cell Explorer allows you to acquire 3D images of your cell cultures in real-time and in a non‐invasive way.

Lung Cancer Cells: 100% Confluency

Human lung cancer cells were grown to 100% confluency in complete DMEM medium (Dulbecco’s Modified Eagle Medium) in 35mm glass‐bottom culture dishes (FluoroDishes™ WPI, #FD35-100).

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