3D Cell Cultures

Nanolive imaging requires samples to be transparent in order for the light to pass through and be collected underneath; thus it is normally suited for high spatio-temporal resolution imaging of live 2D cell cultures. However, this rule of thumb has some exceptions that we will analyse on this page.

3D cell culture systems provide the natural microenvironment of cells, which leads to the obtention of data that is physiologically relevant and more predictive for in vivo tests.

3D cell cultures allow for an improved visualization of cell-cell and cell-matrix interactions. Cell structure and cell populations in 3D cell cultures represent a closer approach to in vivo architecture than traditional 2D monolayer cultures.

Some fields of applications such as drug discovery, stem cell research and cancer cell biology among others are strongly susceptible to benefit from the advantages of 3D cell culture systems.

HeLa cells in gel

Hydrogels are becoming more and more popular as platforms for three-dimensional (3D) cell culturing. 3D 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, 3D 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 3D 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 EPFL.

Immunofluorescence combined with confocal microscopy is one on the most common ways for the study of cells embedded in 3D 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 3D matrix and antibodies could also result in mislabeling, producing a non-specific signal. 

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

Image: a. 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). b. & c. 100 μm-diameter agarose microgels encapsulating mESs (mouse embryonic stem cells). High-throughput combinatorial cell co-culture using microfluidics, 28 Apr 2011, Ethan Tumarkin et al.

Images: 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).

3D alginate beads with E.coli

Nanolive’s 3D Cell Explorer enables accurate and quantitative 4D spatio-temporal monitoring of bacteria cultures.

It allows you to:

  • Monitor several E.coli bacteria colonies growing into 3D systems stain-free (e.g. alginate beads).
  • Monitor the volume of a single bacterium or of a growing bacteria colony
  • Image and discriminate multi-layers of bacteria totally stain-free

Bacteria are tiny, single-cell microorganisms, usually just a few micrometers in length. They may have different shapes, generally spherical (cocci), rod shaped (bacilli) or spiral. Although most bacteria are harmless, several are pathogenic and can cause life threatening diseases such as tuberculosis, tetanus and pneumonia. Other bacteria are instead beneficial and live in symbiosis with other organisms. A typical example is the gut flora. It consists of a complex community of bacteria and other microorganisms that live in the digestive tracts of animals and insects, assisting in digestion.

This video shows E.coli bacteria embedded in alginate beads (generated by Encapsulation Unit – Var J30, 40-70 microns diameter). The beads were mounted on a slide into Minimum Media (MM) diluted 1:4 with distilled water (plus 1% v/v Fumarate 100mM) . The time-lapse imaging experiment was conducted with a standard top-stage incubator set to 37°C and 90% humidity for 3 hours, capturing images every 10 minutes.

Image courtesy of prof. Van Der Meer Jan Roelof (Département de microbiologie fondamentale, UNIL, Lausanne, Switzerland).

This video shows E.coli bacteria (grown overnight in LB medium) that were centrifuged (8000g for 20 minutes) in a tube. The pellet was split between a microscopy slide and the coverslip creating a multi-level bacteria in PBS.

Micro-pillar

Cells on-a-chip

The 3D Cell Explorer lets you analyze morphological changes and membrane remodeling due to functionalized surfaces

Observe how the adhesion of the cell to the substrate is guided by the nanostructures on the device surfaces

  • Determine the differences between cells cultured on a chip with cells on a conventional two-dimensional dish.
  • Study cell-nanostructure interactions using polymeric nanopillars
  • Monitor the focal adhesions formation and the cell-spreading area with fluorescent markers

2D cell culture systems do not accurately recapitulate the structure, function, physiology of living tissues, as well as highly complex and dynamic three-dimensional (3D) 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 getting a non invasive, 3D real time imaging of cells directly on this glass chips.

Nanopillars Glass Chip

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

Collagen matrices

Collagen is the main protein present in the extracellular matrix.

3D cell cultures in collagen matrix mimic in vivo environment. Cell-matrix interactions in 3D cell cultures represent a closer approach to in vivo cell behaviour.

The 3D Cell Explorer allows for high spatio-temporal resolution images of 3D cell cultures over time.

Dendritic cells in Matrigel

This video demonstrates 3D visualization of Inactive and Activated dendritic cells in 3D matrix (Matrigel).