Notes d’applications

Multiplying cryo-electron tomography sample yield through synergistic impact of micropatterning, in-situ fluorescence imaging and ice contamination minimization

In this application note, we demonstrate how the integration of the PRIMO Micropatterning system (Alveole), the METEOR in-chamber cryo-fluorescence microscope (Delmic) and the CERES Ice Shield (Delmic) into the conventional cryo-ET workflow can address the following challenges: cell clusters, cells adhering on the grid bars, in-chamber ice contamination, devitrification and correlation mismatch. We showcase the impact of the upgraded workflow on in-situ correlative cryo-FIB milling of human RPE-1 cells.

HyPE : A Height-Controlled Hydrogel Polymerization Solution

In this application note, we demonstrate the utilization of HyPE, a photopolymerization solution, for the precise and localized control of the height of PEG-based hydrogels to make structures to grow cells in 3D. This approach, in synergy with our versatile bioengineering platform, PRIMO, provides an efficient means to produce hydrogel structures with precise dimensions and shapes. We subsequently apply this methodology to fabricate non-adhesive hydrogel constructs tailored to facilitate the growth of 3D cellular aggregates.

Advancement in EM acquisition by coupling micropatterning with STEM-in-SEM

Discover the game-changing benefits of incorporating PRIMO at the forefront of your STEM workflow and experience a significant reduction in analysis time for electron microscopy. Not only does this enhance efficiency, but it also renders cells more receptive for studying rare intracellular phenomena.

Micropatterning of primary neurons with PRIMO

In this application note, we proposed a full micropatterning protocol widely accessible to neuroscientists. We showed the compatibility of our micropatterning protocol with the Banker culture protocol, very commonly used for primary hippocampal neurons. We demonstrated that we can successfully keep neurons in culture for weeks thanks to the PLPP Gel protocol, allowing analysis to be carried out throughout neuronal development. Altogether, PRIMO’s capabilities to easily create multiprotein patterned substrates or print gradients of proteins open a wide range of possibilities in the development of new in vitro assays in Neuroscience.

Increasing cryogenic electron microscopy experiment output with PRIMO

Cryo-ET provides three dimensional images of biological material in their close-to-living state. Nevertheless, cryo-ET cell imaging experiment has limitations such as data collection throughput. In this application note, we demonstrate the high added value of PRIMO in the workflow of cryo-ET experiment thanks to the precise cell positioning and other advantages that can be of interest for cryo-EM experiments.

Spatially organized and reproducible spheroid formation using PRIMO contactless photopatterning system

High-throughput micropatterning on 96 well plates using PRIMO contactless photopatterning system

Micropatterning of protein is a powerful technology to control the spatial organization of cells and their biochemical microenvironment. We have developed PRIMO: a contactless and maskless UV projection system to facilitate micropatterning experiments. However, a major drawback until recently was the long illumination time that prevented from using PRIMO for high content screening experiments. Here we present a new photo-initiator and protocol allowing fast micropatterning of protein inside standard 96 well plates.

Fabrication of 3D microstructures with a grayscale photoresist using PRIMO maskless and contactless photopatterning technique

The microfabrication of structured substrates by photolithography is a very powerful tool to recreate in vivo-like environments for cell-based experiments. However, the main techniques often use photoresists that only allow the creation of one or two levels in the structure thus limiting the range of shapes. Here, we present the use of a grayscale resist which, combined with PRIMO, a contactless and maskless UV projection system, makes it possible to create much more complex structures than the most common techniques. We further show that it is also possible to functionalize these structures to create a complex environment for cell culture and experiments.

Fast prototyping of organ-on-a-chip with PRIMO

Allowing to mimic highly complex physio-pathological conditions on miniature chip-scale devices, microtechnologies have the potential to revolutionize biology. Among them, microfabrication of polymer materials is of great interest for biomedical applications. However, microfabrication is a complex, multi-step process making the prototyping of a chip for a specific application both time and money consuming. Here, we present PRIMO, a maskless, on-demand UV projection system allowing fast prototyping of microfabricated devices for biological applications.

New Tools for Understanding the Role of the Extracellular Matrix in Cell Morphology: A Combined Photopatterning in Nanotopography Study

In this application note, the differential impact of the chemical and structural components of the Extracellular Matrix (ECM) was investigated and compared to the structure and organization of HeLa cells. Morphology was assessed for cells cultured on micropatterned Fibronectin (FN) protein arrays formed on nanotopographical features. Through this approach, the delineation of the role and importance of each of these extracellular cues in directing cell organization was accomplished. Spatial patterning of ECM proteins has been repeatedly demonstrated to closely control cell and tissue shape and intracellular organization1–5. When FN patterns are formed in the presence of nanoscale physical features, cells will preferentially align to the orientation of the mechanical cues, while the specific transmembrane proteins that involved in cell adhesion will organize based on the FN patterns.

Controlling the topography and biochemistry of cell culture substrates with PRIMO® photopatterning system

In vivo, the cellular microenvironment has a crucial impact on the regulation of cell behavior and functions such as cellular differentiation, proliferation and migration. One of the challenges confronting cell biologists is to mimic this microenvironment in vitro to more efficiently study living cells and model diseases. Here we present PRIMO: a contactless and maskless UV projection system, which allows to control the topography and chemistry of in vitro microenvironments. We first show that PRIMO is a suitable tool to structure photosensitive resists and create molds on which elastomeric solutions (PDMS) can be polymerized. Then we show that the structured PDMS can be specifically functionalized with biomolecules using UV-light structured by PRIMO and a specific photo-initiator (PLPP). Altogether, we demonstrate that PRIMO allows to tailor the cell microenvironment topography through microfabrication and biochemistry through micropatterning.