We are inviting you to join the 2nd edition of our Virtual Symposium, featuring the last publications and results from our users in cell biology and mechanobiology.

Come, watch and interact with them, to understand how they used PRIMO’s micropatterning, hydrogel structuration or microfabrication capabilities to address their experimental issues and improve their in vitro cell models.

Stay after the presentation for a round table on the future of bioengineering, hosted by Benoit Ladoux from Institut Jacques Monod, FR.

Programme of the symposium

2nd Edition – Focus on Alvéole users’ research work in cell biology and mechanobiology

 

TUESDAY, JUNE 7th, 2-5pm CET (Paris time)

Chairman: Matthieu Opitz, Application Engineer at Alvéole

Join this symposium to:

  • get insights directly from your peers on the PRIMO photopatterning technology,
  • investigate recent research developments achieved in your research field,
  • have the opportunity to ask the speakers all the questions you have in mind!

Hydrogel patterning techniques for applications in traction force microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. We present a protocol to rapidly and efficiently fabricate micropatterned polyacrylamide hydrogels for TFM studies. The micropatterns are first created by maskless photolithography using near-UV light, where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is realized by controlled activation of aldehyde groups, resulting in adhesive regions of desired shapes to accommodate either single cells or groups of cells. For TFM measurements, we use dual-layered polyacrylamide hydrogels of different elasticity, and we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells.

Ada Cavalcanti is currently a group leader at the Max Planck Institute for Medical Research in Heidelberg and Member of the Faculty of Biosciences at the Heidelberg University. Her research is centered on the mechanobiology of receptor-mediated cell adhesion. She completed her Master studies at the University of Pennsylvania, USA and then obtained a PhD in Biosciences in 2005 at Heidelberg University, Germany. She spent her postdoctoral years at the Max Planck Institute for Intelligent Systems until she started her group in 2011. She has been awarded in 2008 with the prize “for women in science” from UNESCO-L’Orèal.

 

1D micro-nanopatterned integrin ligand surfaces as tool to modulate cell front and back coordination for directed migration

Cell-extracellular matrix (ECM) adhesion modulated by integrin receptors is a highly regulated process involved in many vital cellular functions such as motility, proliferation and survival. However, the influence of lateral integrin clustering in modulating the cell front and back dynamics during cell migration remains unresolved. Therefore, we developed a 1D micro-nanopatterned migration protocol based on the block-copolymer micelle nanolithography (BCMNL) technique with biofunctionalized gold nanoparticles with the integrin-specific RGD (arginine-glycine-aspartate) motif. Defined 10 mm-wide micropatterned stripes were fabricated consisting of a quasi-perfect hexagonal arrangement of gold nanodots with a mean diameter of 8 nm that serve as an anchoring site for a single integrin heterodimer. The gold nanoparticles were placed with a lateral spacing of 50, 80 and 100 nm to regulate integrin clustering and focal adhesion dynamics. By employing time-lapse microscopy and immunostaining, we propose that the speed, coordination of front-back dynamics and migratory behavior of fibroblasts change according to the nanoscale spacing of adhesion sites.

Victoria Levario Diaz is currently a postdoctoral researcher in the Cavalcanti-Adam group at the Max Planck Institute for Medical Research in Heidelberg. She completed her Master studies and obtained her PhD in Nanoscience in 2021 from the University of Bristol, UK. She joined the Cavalcanti-Adam group in the fall of 2020 to develop protocols combining nanoscience and micropatterning to direct and analyze cell migration.

Bioengineering a Miniaturized In Vitro 3D Myotube Contraction Monitoring Chip

Using the PRIMO system from Alvéole, our team has recently developed a microfabricated platform to produce 3D muscle organoids suspended between two flexible pillars. Optimized micropatterned substrate design enabled to obtain high culture yields in tightly controlled microenvironments. The technology allows monitoring of the forces developed during spontaneous contractions of the obtained microtissues, based on in situ observation of pillars deflection. The developed system also enables to recapitulate pathological phenotypes, as was reported for LMNA-related Congenital Muscular Dystrophy (L-CMD) modeling, with successful development of mutant 3D myotubes displaying contractile dysfunction. Importantly, the system requires significantly less patient material than other 3D-microtissues approaches, which has the potential to substantially improve drug screening capability.

Léa Trichet has a background in biophysics and medicine and carried out her doctoral work on the topic of cell movement biomimetism at Institut Curie, under the supervision of Cécile Sykes and Julie Plastino. She pursued her academic career in the group of Benoît Ladoux, working on cell-matrix interactions, and further worked for General Electric Healthcare as a Global Clinical Leader in Interventional Radiology, to translate clinical needs into products. She was appointed CNRS researcher in 2014 in the MatBio team at the LCMCP, Sorbonne Université. She works on the physical chemistry of biopolymers to develop biomimetic musculo-skeletal models and new biomaterials for tissue regeneration. Her research also focuses on the development of “tissue on chip” devices offering controlled microenvironment to monitor contraction of muscular microtissues. Since 2019 she also works as a mission head for innovation and industrial partnerships at the Institute of Biological Sciences, CNRS.

Lab on a chip for crowd experimental studies

Studying crowd movement is becoming a major societal issue since, with demographic growth, we are also witnessing an increasing demand for individual mobility. Institutions must therefore organize themselves to manage the large flows and gathering of people. This societal issue leads, first, to a theoretical challenge, as it involves building new statistical models to simulate crowds. This brings an experimental issue: how to set up experiments in the laboratory, using living active matter, to test the robustness of these theoretical models?
We propose to create a lab on a chip combining photolithography and living cells to model crowd movements. Keratocyte epidermal cells, extracted from fishes, are outstandingly fit for this task due to their exceptional motility. These cells are migrating from scales on a coverslip, by healing reflex. On this coverslip, micro-environments of adhesive proteins have been created using photolithography. It allows us to burn a specific region and to create the desired geometries. We can therefore multiplex the experiments, obtain quantitative datas and play on the geometries to infinity.
Abstracting this idea further, we imagined it would be an interesting task to compare the navigation of cells in the micro-world with the movement of living agents in the macro-world. We already obtained a proof-of-concept that this experimental setup can fit for humans moving in an escape scenario through a bottle-neck. It has been proved that when the desired velocity of pedestrians is increasing beyond a critical point, jams emerge around the exit, leading to lower flow rate and a ‘stop-and-go’ regime, where pedestrians move forward in intermittent waves. The same phenomenon has been observed on cells, in the same architecture at the right scale. Also, by displaying residence time and density maps, on human and cell experiments, we obtained very nice analogies, promising for the future of this project. A huge advantage of our system is the facility we have to control a whole bunch of parameters, allowing us to refine our study, and to play on the density and the initial speed of the cells, as it is done on humans with instructions on how they must behave.
The analogy could be taken further, by extending the studies to humans moving in urban spaces or vehicles navigating in cities. Contrasting the motility of cells with the movement of animals, each living in their own unique spatial and temporal scales, may just be the key to extracting universal insights into the dynamics of active matter systems.

Martial Balland is a biophysicist from the Laboratoire Interdisciplinaire de Physique- LIPhy (CNRS/UJF Grenoble), who tries to understand biological processes through physical approaches.

In vitro assembly and tension formation of a reconstituted sarcomere

The generation of force in skeletal and cardiac muscle is achieved by contractile arrays of actin and myosin filaments. These fibers are arranged in repeating subunits called sarcomeres and constitute the smallest functional unit capable of converting chemical energy into muscle movement. Due to their complex regulation and limitations set to experimental studies in cells, we propose a minimal reconstructed sarcomere to enable a biophysical characterization of the self-assembly, as well as the tension formation of fibers found in sarcomeres. By using a micropatterning technique, we print actin binding proteins to mimic the linear array of actin bundles in sarcomeres. We elucidate how different isoforms of non-muscle myosin 2 (NM2) organize these bundles in vitro, resembling mature myofibrils found in cells. Finally, we show how NM2 builds tension in these structures, leading the way to quantify the molecular basis of muscle force production.

Philip Bleicher was trained as Molecular Biotechnologist at the Technical University in Munich (TUM). For his PhD he moved to the Physics Department (TUM) to study the dynamics of the actin cytoskeleton in the group of Cellular Biophysics of Prof. Andreas Bausch. After completing his thesis and obtaining a fellowship at the NHLBI at NIH, he moved to the United States and continued his research in the Molecular Physiology lab of Dr. James Sellers.

PRIMO bioengineering technology allows the reconstruction of functional intrahepatic bile ducts with predefined geometry

Building bile ducts is a challenging task for liver tissue engineering, in particular the reconstruction of the intrahepatic biliary tree consisting of 10-100µm ducts. Although several methods were used to address such a challenge, the resulting structures either had luminal diameter corresponding to the extrahepatic part of the tree or had unpredictable geometry [1]. We therefore developed a biocompatible stereolithographic approach to encapsulate cholangiocytes, the biliary epithelial cells in 3D structured hydrogels mimicking the branched biliary network [2]. This strategy allowed us to produce functional Biliary Structures by PhotoPolymerization (BSPPs) with a predefined geometry and with a final luminal diameter at the intrahepatic duct scale [3].

[1]Lewis, P. L. et al. Sci. Rep. 8, 12220 (2018).

[2] Ma, X. et al. Proc. Natl. Acad. Sci. 113, 2206 (2016).

[3] Mazari-Arrighi, E. et al. Biomaterials. 279, 121207 (2021).

This work received the financial support of the iLite RHU program (grant ANR ANR-16-RHUS-0005)

Elsa Mazari-Arrighi holds a PhD in biophysics from Paris-Sud University and is specialized in development of 3D architectures for cell biology.  Since 2017, she is a postdoctoral researcher at Hospital St Louis in a joint research unit (U976 Stem Cell Technologies, Inserm/ Université Paris Diderot) and she focuses on the development of 3D bioprinting approaches to reconstruct functional tissues.

Pattern-Based Contractility Screening, a Reference-Free Alternative to Traction Force Microscopy Methodology

The sensing and generation of cellular forces are essential aspects of life. Traction force microscopy (TFM) has emerged as a standard broadly applicable methodology to measure cell contractility and its role in cell behavior. While TFM platforms have enabled diverse discoveries, their implementation remains limited in part due to various constraints, such as time-consuming substrate fabrication techniques, the need to detach cells to measure null force images, followed by complex imaging and analysis, and the unavailability of cells for postprocessing. Here we introduce a reference-free technique to measure cell contractile work in real time, with commonly available substrate fabrication methodologies, simple imaging, and analysis with the availability of the cells for postprocessing. In this technique, we confine the cells on fluorescent adhesive protein micropatterns of a known area on compliant silicone substrates and use the cell deformed pattern area to calculate cell contractile work. We validated this approach by comparing this pattern-based contractility screening (PaCS) with conventional bead-displacement TFM and show quantitative agreement between the methodologies. Using this platform, we measure the contractile work of highly metastatic MDA-MB-231 breast cancer cells that is significantly higher than the contractile work of non-invasive MCF-7 cells. PaCS enables the broader implementation of contractile work measurements in diverse quantitative biology and biomedical applications.

Ajinkya Ghagre, a PhD student in the Department of Bioengineering at McGill University, is a biophysicist interested in studying the role of physical forces and mechanics in regulating cell biology. He designed and developed PaCS (Pattern-based Contractility Screening), a novel tool to simplify cell contractile force measurements and utilized it to quantify contractile force changes in breast cancer cells. In the future, he aims at upgrading this tool into a high throughput cell contractile screening technology which will be used for drug validation and can potentially cut the cost for drug discovery process.

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