Just before the Christmas holiday break we had the PhD dissertation defence of José. He explains his work developing “Sandwich-like systems to engineer the cellular microenvironment” in this post.
José Ballester-Beltrán, PhD student at MiMe to December 2014.
When I first met Prof. Manuel Salmerón-Sánchez, we discussed about the key role that 3D culture systems were going to have for the Tissue Engineering and Cell Biology fields. Indeed cell biology is still commonly studied on 2D substrates such as petri dishes or tissue culture plates. These differ to the in vivo 3D environment in terms of dimensionality, stiffness and other parameters. Consequently, cells cultured on these substrates have to adapt to this unnatural (flat and stiffer) environment resulting in unreliable behaviours. Hence, when a culture condition is tested in vitro under this culture environment (such as the efficacy of a drug), we cannot be sure whether the results are due to the drug or the adaptation of the cells to this flat environment; and most importantly, we cannot assure a similar result in vivo.
Nowadays we all expect more useful results when the culture system mimics the physiological environment of the specific cell type. Consequently there is now a wide variety of 3D systems that provide environments closer to the physiological and allow the study of cell biology under more reliable conditions.
However, working with 3D systems is not easy:
– 3D systems are usually more difficult to handle. They are easy to break or denature, and the protocols are not always easy to perform.
– Current experimental procedures on 2D substrates cannot be easily established for 3D cultures. Particularly relevant are the difficulties in imaging a whole 3D system, or the extraction of proteins & nucleic acids from a 3D culture.
– It is difficult to have a reliable 2D control to understand the role of the dimensionality in cell behaviour.
In addition, the high variability and complexity of 3D systems hinders their standardization in common cell culture procedures. Consequently we wondered whether we could develop a simple culture system that could overcome some of these disadvantages while mimicking physiological environments.
We started our work from the statement that cell-protein-material interaction is a key process in cell biology since this triggers signalling pathways that modulate important cellular processes. In 3D systems this interaction occurs all around the cell while on 2D substrates it only occurs on the ventral side (where cells are attached to the substrate), becoming therefore an interesting feature that could be able to induce the behavioural differences. We thought that a sandwich-like system based on a 2D configuration could be able to modulate the cell-protein-material interaction in a 3D-like way and at the same time could be a useful system to understand differences with the traditional 2D substrate.
The basic description of a sandwich-like system for cell culture is that, once having the cells adhered on a 2D substrate, a second 2D substrate is overlaid on top of the cells in order to trigger the excitation of the dorsal receptors. Consequently cells interact ventrally and dorsally with the substrate and thus response alike 3D environments.
Due to the nature of the system, sandwich-like culture is a simple and versatile tool that allows the study of different parameters in cell/material interactions such as chemistry, topography, stiffness and protein coatings at both the ventral and dorsal sides since one only need to change the type of substrate. It provides a higher degree of versatility compared to other 3D systems due to the independent dorsal and ventral combination of a wide variety of surface conditions. Additionally, the role of this dorsal stimulation can be easily compared to the traditional 2D systems.
In order to overcome some of the issues related to the 3D systems, poly-L-lactic acid (PLLA) and poly(ethyl acrylate) films were used as dorsal substrates since they are transparent and allow microscopic procedures. Besides, since this materials are not degradable, cells are constrained to move within the same plane, easing cell tracking.
Protein and nucleic acid extraction can be easily performed by simply opening the sandwich-like culture in order to lyse cells.
We have shown cells cultured within the sandwich-like system undergo important changes with respect to 2D cultures and behave similarly as within 3D systems in terms of cell morphology, proliferation, differentiation and migration. These results corroborate therefore the key role of 3D cell/material interaction on cell fate. To sum up, sandwich-like culture is a simple system that offers the possibility to mimic different 3D-like microenvironments to investigate cell fate.
Footnote: JoVE has accepted a paper, soon to be published, describing in detail (with videos!) the protocols devised to work with sandwich-like cell cultures. The paper is authored by José Ballester-Beltrán, Myriam Lebourg and Manuel Salmerón-Sánchez.