Microscopy & Microtechniques Micropatterning: The art of micro-controlling cells
Marie-Charlotte Manus* (International Marketing Manager), Dr Louise Bonnemay (Research Scientist) and Matthieu Opitz (Research Scientist), Alvéole, 30 rue de Campo Formio 75013, Paris, France
*Corresponding author
marie-charlotte.manus@
alveolelab.com The challenge of studying living cells in vitro
Like all living beings, cells are sensitive to their environment which infl uences their behaviour and development (cell differentiation, proliferation, migration, etc.). Indeed, several studies have shown that mechanisms such as cancerous growth or stem cell differentiation are not only induced by genetics but also by the cellular environment [1,2]. However, its complexity in terms of biochemistry, structure or interactions explains the diffi culties faced when studying living cells in vitro. In addition to this problem of physiological relevance, researchers are faced with many other challenges, such as the recurring reproducibility issues, ease of use and effi ciency, even more true in the case of high throughput.
techniques have been developed and improved over the years, fi rst by academic research labs and then by industrial companies to broaden its access, facilitate its use and guarantee reproducible results. Nowadays, the two most commonly used micropatterning approaches are microcontact printing using elastomer stamps and deep UV photolithography through photomasks [6]. These approaches however lack some capabilities now often considered mandatory, including fl exibility in pattern shapes, multi-substrates compatibility (due to a growing use of soft or micro structured substrates) and alignment on these substrates; these limitations constrained the use of micropatterning to plain fl at substrates for a long time.
Furthermore, the increased complexity of cell biology experiments caused an increased diversity of substrates with an increased fragility, due to their material composition, low stiffness or microscopic size. A typical example of that fragility would be the electron microscopy grids (EM grids) used for whole-cell cryo-electron tomography [7] - a circular substrate about 3 mm diameter, composed of a metallic mesh grid and a carbon layer fi lm approximately 20 to 50 nm thick.
Figure 1. Researcher checking on cells in culture media. Credit: Leica Microsystems
Among the experimental tools and techniques used to solve these issues is micropatterning: a method routinely used in mechanobiology, known as the in vitro analysis of living cells intra and inter-cellular mechanisms. But far from being limited to mechanobiology, micropatterning can be a powerful asset for different fi elds of research, such as disease modelling, immunology, toxicology. It also recently appeared as a must- have for the promising fi eld of cryo-electron tomography (cryo-ET): a technique based on cryo-electron microscopy (cryo-EM) which allows the imaging of macromolecular complexes in fl ash frozen cells and therefore a better understanding of their role in their native state.
This article will investigate the benefi ts of micropatterning in cell biology studies and then for the emerging fi eld of whole-cell cryo-ET.
Micropatterning is defi ned as the controlled deposition of biomolecules at the micron scale aiming to mimic or control the cell biochemical microenvironment. It was fi rst used in the cells biology research about 30 years ago to investigate the link between cell shape (using adhesive proteins to locally control cell adhesion and shape) and terminal differentiation [3]. Since then, micropatterning has been routinely used to demonstrate the link between cell adhesion, cell shape and intra-cellular organisation [4,5].
Different micropatterning
Recently, one micropatterning solution seemed to answer all these issues of design fl exibility, alignment and surface preservation by preventing direct contact with the substrate. This innovative system is the PRIMO®
maskless photopatterning system
developed by Alvéole, a French spin-off from Centre National de la Recherche Scientifi que, France (CNRS), funded in 2010, the mechanism of which will be presented in this article among others.
Micropatterning workfl ow:
A combination of repulsion and attraction All protein micropatterning techniques rely on the use of both adhesive patches (micropatterns), formed with adhesive proteins such as fi bronectin or laminin, and repellent areas generated thanks to an anti-fouling coating. The quality of the anti-fouling coating – classically a polyethylene glycol (PEG) polymer – is critical to ensure the quality of the protein and cell micropatterning, in terms of longevity and shape accuracy. Indeed, when seeded, the cells should not be able to adhere to the repellent surface but only to the micropatterns.
Examples of micropatterning techniques include: 1. Microcontact printing
Figure 2. RPE1 cells (nucleus in blue, actin in red) adhering to fi brinogen micropatterns (green) of different shapes. Credit: Alvéole
For this approach, a stamp with the desired microfeatures must be microfabricated using photolithography, photomasks and photoresists to generate a master displaying the designs of the future micropatterns. Polydimethylsiloxane (PDMS) is then poured on to the master and cured to create the stamp (opposite replicate of the initial master). The ‘ink’ for this stamp is a solution of adhesive protein. The non-patterned areas are then backfi lled with an antifouling layer. This micropatterning technique is the one used most frequently and also considered the easiest (if buying the stamp from a provider instead of fabricating it in-house), but it is also the least precise one. Indeed, the sharpness of the stamp’s pillars edges, the pressure exerted on the stamp and the difference in protein quantity deposition, are all variable factors that ultimately affect the quality of the fi nal micropatterns. And of course, at the microscopic scale,
INTERNATIONAL LABMATE - NOVEMBER 2020
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