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.

Mesenchymal stem cell interacting with 3D hydrogel

Stem cell therapies hold widespread hope for the treatment and/or prevention of various degenerative diseases. There is thus, widespread interest in understanding what controls their differentiation into specialized cell types. The local microenvironment surrounding the cell, the “stem cell niche”, has long been recognized as being critical to the differentiation process1, however early studies focused on the importance of soluble factors rather than bio-physical signals.

Over the past decade, researchers have begun to appreciate the importance of mechanical signalling in the stem cell differentiation process2. We now know that stem cells can sense and respond to many biophysical cues in the niche including matrix elasticity, topography, shear stress and strain forces2. Matrix elasticity (the stiffness of a surface) in particular, has generated great interest in the field of regenerative medicine, as it has been shown to play a critical role in determining stem cell fate3,4. The goal now, is to replicate what cells experience in vivo to improve stem cell culture in vitro

3D polyethylene glycol (PEG) hydrogels have shown great promise in this respect5. Here we tested whether PEG hydrogels are compatible with our 3D Cell Explorer. We grew mesenchymal stem cells in 3D PEG hydrogels of varying degrees of stiffness, which were kindly provided by Dr. Sonja Giger and Prof. Matthias Lutolf from EPFL, Switzerland. We then imaged cells for 48 hours at an acquisition rate of one image every 30 seconds. A post-processing colour scale was applied to data in the z-axis, to add spatial information to our images. Blue indicates where the cell is closer to the surface of the gel, while pink indicates where it is further away.

The solid gel had no impact on image quality; cells spread further and were more mobile on soft gels than stiff gels but were equally healthy on both. Images taken of cells on one of the soft hydrogels were particularly interesting. The surface of the gel in question had slight indentations on the surface creating a more complex nanotopography. Although not the intended aim of the experiment, the video of this gel (shown here) highlighted some of the interesting interactions that occur at the surface interface. The cell can be observed pulling and pushing on gel pieces and occupying the holes on the surface (we marked these moments in the video with circles). These observations show that the combination of 3D hydrogels and holotomography can open the door for novel observations to be made about how stem cells sense and interact with their physical environment.

For more information or to organize a demo in your lab please click here

References

  1. Scadden, D. T. The stem-cell niche as an entity of action. Nature 441, 1075–1079 (2006).
  2. Sun, Y., Chen, C. S. & Fu, J. Forcing Stem Cells to Behave: A Biophysical Perspective of the Cellular Microenvironment. Annu. Rev. Biophys. 41, 519–542 (2012).
  3. Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix Elasticity Directs Stem Cell Lineage Specification. Cell 126, 677–689 (2006).
  4. Huebsch, N. et al. Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate. Nat. Mater. 9, 518–526 (2010).
  5. Nam, S., Stowers, R., Lou, J., Xia, Y. & Chaudhuri, O. Varying PEG density to control stress relaxation in alginate-PEG hydrogels for 3D cell culture studies. Biomaterials 200, 15–24 (2019).

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).