Alvéole symposium: Focus on our users research work

Towards exposing the forces remodeling nematic 3D tissue protrusions by combining micro-patterning and soft hydrogel pillars

Cells continuously generate forces in order to move and deform. In tissues, cells need to coordinate their activity to generate the long-ranged flows and forces able to remodel tissues, including transformations in shape and function [1]. Detection of these forces has been possible in vivo by introducing deformable, passive, objects in tissues [2,3]. However, these technologies are not applicable to experiments based on cell monolayers, with better control capabilities. Here, I present how PRIMO has allowed us to prepare (1) robust cell-adhesive micro-patterns, able to confine cells for weeks and secure the formation and stability of 3D tissue protrusions, and (2) micro-systems, composed of both patterns and soft hydrogel pillars, with which we are able to reveal and quantify the forces remodeling our cellular system, from nematic cell monolayers to minimal 3D tissues.

[1]      P. Friedl, Y. Hegerfeldt, and M. Tusch, Collective Cell Migration in Morphogenesis and Cancer, International Journal of Developmental Biology.

[2]      O. Campàs, T. Mammoto, S. Hasso, R. A. Sperling, D. O’Connell, A. G. Bischof, R. Maas, D. A. Weitz, L. Mahadevan, and D. E. Ingber, Quantifying Cell-Generated Mechanical Forces within Living Embryonic Tissues, Nat. Methods 11, 183 (2014).

[3]      M. E. Dolega, M. Delarue, F. Ingremeau, J. Prost, A. Delon, and G. Cappello, Cell-like Pressure Sensors Reveal Increase of Mechanical Stress towards the Core of Multicellular Spheroids under Compression, Nat. Commun. (2017).

[4]      P. Guillamat, C. Blanch-Mercader, K. Kruse, and A. Roux, Integer Topological Defects Organize Stresses Driving Tissue Morphogenesis, BioRxiv 2020.06.02.129262 (2020).

Pau Guillamat received his bachelor’s degree from the University of Barcelona, where he did his PhD on Active Matter in the group of Self-Organized Complex Materials, under the supervision of Drs. Jordi Ignés and Francesc Sagués. There he focused on developing strategies for controlling the dynamics of active gels. Then Pau moved to Geneva to work in the lab of Dr. Aurélien Roux to study self-organization principles inducing remodeling within cell monolayers.

Investigating cellular responses in multi-cue environments using multi-dimensional protein patterning

The extracellular microenvironment is an important regulator of cell function. Numerous structural cues present in the cellular microenvironment, such as ligand distribution and substrate topography, have been shown to influence cell behavior. However, the roles of these cues are often studied individually using simplified, single-cue platforms that lack the complexity of the three-dimensional, multi-cue extracellular matrix cells encounter in vivo. To bridge this gap, our group combines optics-based protein patterning afforded by Primo technology and lithography-based substrate microfabrication, which enables high-throughput investigation of complex cellular environments. We created patterns of extracellular proteins (resembling contact-guidance cues) on a 2.5D cell culture chip containing a library of well-defined microstructures (resembling topographical cues in tissues). We will discuss how this unique approach reveals a variety of intriguing cellular phenotypes and cell-type-dependent responses that are governed by a complex interplay between the dynamics of actin stress fiber (re)organization and force generation, nucleus mechanics, and adhesion morphology. These findings exemplify the potential of this approach for systematic investigation of cellular responses to multiple microenvironmental cues.

Dr. Nicholas Kurniawan is an Assistant Professor at the Department of Biomedical Engineering in Eindhoven University of Technology, the Netherlands. His research focuses on understanding how the physical and mechanical interactions between cells and cellular environments shape physiological tissue function and drive pathologies. His interdisciplinary team combines approaches from biophysics, mechanobiology, microfabrication, and cell biology with an outlook of exploiting cell–materials interactions for regenerative medicine applications. His work is supported by grants from the European Research Council, the Dutch Research Council, the Dutch Ministry of Economic Affairs and Climate Policy, and the Dutch Ministry of Education, Culture and Science.

Photopatterning and microfabrication to generate vascular in vitro models

D.G. Kasi1,2, M.D. Ferrari2, A.M.J.M. van den Maagdenberg1,2 and V.V. Orlova3 Departments of 1Human Genetics, 2Neurology, and 3Anatomy and Embryology, Leiden University Medical Center, Leiden, the Netherlands

To develop vascular in vitro models, our group utilizes the Alvéole PRIMO system combined with human induced pluripotent stem cells (hiPSCs). Our in-house developed hiPSC-derived vascular cells can be photopatterned with PRIMO to produce arrays of cells with predefined morphology. We seek ways to perform standardized high-throughput contraction screening of hiPSC-derived vascular smooth muscle cells (vSMCs).
Additionally, we employ microfabrication to produce vascular contractile tissues. In collaboration with TU Delft we are developing a novel actuation/sensing material that is able to both sense tissue contraction and generate cyclic strain. We use PRIMO for microfabrication to rapidly prototype microgrooved PDMS substrates that align our vSMCs to induce anisotropic properties. The resulting vascular contractile tissue can be used in combination with the novel actuation/sensing material for compound screening based on the contractile force of the tissue.

Dhanesh’s research is focused on the development of vascular in vitro models that support migraine-relevant and vasoactive compound screening. Such models will pave the way for future full-fledged migraine-on-chip models and help understanding pathophysiological processes in other vascular disorders.

Mechanical properties of collective cellular assemblies.

Epithelia are communities of epithelial cells with close intercellular communications and of highly ordered coordination in their motility. Tissues can adjust their internal contractile stresses and organization in response to different stimuli, leading to distinct dynamics. Mechanical properties of epithelial tissues are important for our understanding in many vital biological processes, including homeostasis, morphogenesis, and metastasis and are tightly regulated by cell-cell interactions. I will present two examples highlighting the importance of cell-cell junction mechanosensitivity. In the first part, I will discuss the emergence of collective modes of cell migration based on mechanical signals, cell polarity and cell-cell interactions. In the second part, I will focus on the active nematic properties of cellular monolayers and how this framework can be used to study multicellular biological systems. I will first describe the example of cell extrusion of apoptotic cells from epithelial tissues. I will then discuss why cellular monolayers can display various active behaviors as exemplified by the contractile nature of fibroblasts and the extensile nature of epithelial cells or neural crest cells. Through a combination of experiments and in silico modeling, I will show that cell-cell interactions, stresses transmitted at cell-cell junctions and cell-substrate interface could determine the active nature of multicellular systems.

Benoit Ladoux is a physicist by training, working on cell mechanics. Starting in the single molecule field, he developed a research activity in cell mechanics and adhesion in the laboratory Matière et Systèmes Complexes. In 2008, he was involved in the creation of the Mechanobiology Institute (MBI) directed by MP. Sheetz in Singapore. After spending two years in Singapore between 2010 and 2012, he came back to Paris and joined the Institut Jacques Monod as a senior group leader together with a cell biologist, RM. Mège. In 2015, he moved from a faculty position to the CNRS as a research director. From 2012 to 2018, he shared his time between Paris and Singapore. His research aims at understanding how cell-adhesion mechanisms are associated to mechanotransduction and driven by the mechanical properties of the cellular environment and how mechanosensing regulates cell behaviors and tissue homeostasis.  He developed various tools to analyze the mechanical responses of cells to the physical properties of the environment including rigidity and topography sensing. He studied the impact of substrate stiffness, geometry and curvature on single and collective cell migration. His recent research focused on the impact of mechanics on epithelial homeostasis including cell proliferation and cell extrusion.

The Toxoplasma acrobat microbe: combining biophysics and real time imaging to decode top gliding performance

Cell migration is central to the life of not only multicellular organisms but also single-celled microorganisms including Toxoplasma gondii, the unicellular eukaryotic parasite that belongs to the phylum of Apicomplexa.  T. gondii is a highly polarized few micron-sized cell endowed with unique high-speed gliding motility mode that powers navigation through all tissues of the homeothermic hosts, but also cellular barrier crossing and entry into a hosting cell where to produce progeny. Therefore, the team investigates how forces tune the motile and invasive vital motions for T. gondii survival and expansion in the host. Through collaboration with biophysicists, we have applied time-resolved force microscopy to (i) decipher at the millisecond scale and on sub-micrometric dimensions the contact zones between the parasite and a surface, and (ii) measure the forces in these areas that would account for the typical parasite helical gliding style.  Identifying a contact area between the anterior region of the parasite and the surface at the onset of each motion sequence, we next used PRIMO to design well-demarcated micropattern areas, and established the strict necessity of a pulling force exerted by the parasite from this apical anchoring point. The traction imposes the forward curvature of the parasite which coincides with the energy charge in a spring system enabled by a spiral cytoskeleton formed of microtubules.

The Pavlou et al. has been published in the journal ‘ACS Nano’, with the movement of Toxoplasma visible in a short video produced by the journal (https://youtu.be/kG146bHn4qw).

Isabelle Tardieux is a CNRS research director who currently heads a team working on « Biomechanics of Host-Parasite Interactions” at the Institute for Advanced BioSciences (Cnrs UMR 5309 – Inserm U 1209 – Univ. Grenoble Alpes) in Grenoble (France). She obtained a PhD in Population Dynamics and Entomology before being appointed as assistant professor at the Pasteur Institute (Paris, France) working on vector-borne diseases. She has been successively trained in Cell biology and Parasitology at Yale University (Infectious Diseases Dept, USA) and at the NIH Bethesda (Parasitic Diseases Laboratory, USA) as post-doctoral fellow and visitor associate, respectively. Building on her experience in membrane and cortex dynamics and in live imaging, she has recently broadened her team framework towards biomechanics and nanobiology in collaboration with the laboratory of interdisciplinary Physics (LIPhy) at Grenoble. Email: Isabelle.tardieux@inserm.fr

Pericellular protective microgels to reduce radical damage during lithographic fabrication of cell-laden hydrogel structures

Contributing authors to the study: Laura Hockaday Kang, Lena Prange, Johannes Fels, Christian Schuberth, Roland Wedlich-Söldner

Photoinitiator radicals and their byproducts damage cells in multiple ways, limiting the viability of tissue engineering strategies that use photo-crosslinking hydrogels formed via free-radical-initiated polymerization. There exist few protective strategies that can be broadly applied for multiple cell types during fabrication of cell-laden, hydrogel-based artificial tissue. In this study, we tested the hypothesis that applying a collagen microgel around a cell protects the cell pericellularly against photoinitiator radical damage.  Using a microfluidic flow focusing device, microgels were applied to cells, and then the microgel-treated cells and control (untreated) cells were exposed to activated photoinitiator radicals. Cells were assessed for plasma membrane (PM) integrity using propidium iodide staining. Additional symptoms of damage, such as disruption of actin dynamics, were assessed using Lifeact-GFP. Microgel treatment significantly reduced photoinitiator radical-induced PM compromise and actin disruption in the 3 cell lines tested. To further test the consequences of microgel treatment, microgel-protected cells were incorporated into photopatterned methacrylate gelatin hydrogels.  Using a PRIMO digital micromirror projection platform, light was projected into microfluidic channels to solidify a mixture of microgel-protected cells, methacrylated gelatin, and photoinitiator. Patterned hydrogel composite structures were then cultured and cells within assessed for damage.  Altogether, these results support that microgel treatment could be a feasible protective strategy for cells in fabrication strategies that use photocrosslinking.

Laura Hockaday Kang is a researcher in the Institute of Cell Dynamics and Imaging at the University of Münster, Germany. She received her B.Sc. in Bioengineering at the University of California Riverside in the United States. Before graduate school, Laura worked in a research and development company, Energy Related Devices Inc. in Los Alamos New Mexico, which uses nature-inspired engineering solutions to address technological problems. She received her PhD and M.Sc in Biomedical Engineering at Cornell University, focusing on 3D printing technologies to develop native heterogeneity in tissue engineered heart valves. Her research interests are targeted at understanding what happens to cells in bio-fabricated hydrogel environments and how cells behave and function in engineered tissue

Mechanical forces in stem cell fate regulation

Cells are constantly exposed to a spectrum of mechanical cues, such as shear stress, compression, differential tissue rigidity, and strain, to which they respond to by engaging mechanisms of mechanotransduction. These forces function as important morphogenetic ques that are transmitted to the nucleus to alter genetic programs. On the other hand, excessive mechanical stresses have the potential to damage cells and tissues. Our recent research illustrates how mechanical forces impact stem cell fate and how dynamic changes in chromatin organization in response to force alter the mechanical properties of the nucleus and chromatin to prevent damage, as well how cells can adapt to differential force environments by changing their structure and fate.

Kate Miroshnikova received her Bachelors of Science in Engineering from F.W. Olin College of Engineering in 2009. She then joined UC Berkeley-UCSF Graduate Program in Bioengineering and got her PhD in 2015 in the field of matrix mechanobiology of cancer in the laboratory of Dr. Valerie Weaver at UCSF in San Francisco, USA. After her postdoctoral tenure as a Whitaker postdoctoral scholar in Grenoble, France in the lab of Dr. Corinne Albiges-Rizo studying junctional mechanics, Kate joined the laboratory of Dr. Sara Wickström at the Max Planck Institute for Biology of Ageing in Cologne, Germany as a HFSP and EMBO postdoctoral fellow in nuclear and chromatin mechanics. In 2021 Kate joined the NIH in Bethesda, USA to start her own group in nuclear mechanogenomics. Her research aims to understand how cells sense and integrate mechanical and chemical information from their environment to control cell state and behavior. She is particularly interested in how the nucleus responds to these mechanochemical signals to alter chromatin architecture and gene expression. The research is highly interdisciplinary across the fields of biology, physics, and medicine.

The strange cases of Leukocyte migration and adhesion: swimming and reverse haptotaxis

Upon recruitment from blood to inflamed zones, leukocytes cross blood vessels endothelium at transmigration portals enriched in  integrins ligands¹ and later migrate in inflamed tissues with an integrin-dependent manner^2,3. These observations support that integrins play a crucial role in leukocyte migration and guidance in vivo. Concerning migration, integrin-mediated adhesion is reported to be dispensable in a matrix but not in absence of confinement^3,4, and concerning guidance there exist no direct proof of integrin-mediated haptotaxis with leukocytes yet. Here, we use in vitro protein patterning to show that crawling lymphocytes can swim in suspension without adhesive contacts with a matrix⁵ and that integrins mediate lymphocytes haptotaxis, either with or against a gradient of adhesion⁶.

1 Carman CV, Springer TA. A transmigratory cup in leukocyte diapedesis both through individual vascular endothelial cells and between them. J. Cell Biol. 2004;167(2):377–388.

2 Overstreet MG, Gaylo A, Angermann BR, et al. Inflammation-induced interstitial migration of effector CD4+ T cells is dependent on integrin αV. Nat. Immunol. 2013;14(9):949–958.

3 Lämmermann T, Bader BL, Monkley SJ, et al. Rapid leukocyte migration by integrin-independent flowing and squeezing. Nature. 2008;453(7191):51–55.

4 Reversat A, Gaertner F, Merrin J, et al. Cellular locomotion using environmental topography. Nature. 2020;582(7813):582–585.

5 Aoun L, Farutin A, Garcia-Seyda N, et al. Amoeboid Swimming Is Propelled by Molecular Paddling in Lymphocytes. Biophys. J. 2020;119(6):1157–1177.

6 Luo X, Noray VS de, Aoun L, et al. Lymphocytes perform reverse adhesive haptotaxis mediated by LFA-1 integrins. J. Cell Sci. 2020;133(16):.

Olivier Theodoly is a CNRS research director working as a biophysicist at the “Laboratoire Adhesion & Inflammation” (Inserm/CNRS/Aix Marseille université). He is currently studying the properties of immune cell recruitment in vitro using reductionist approaches based on microscopy, microfluidics and micropatterning.

Cytoskeletal dynamics in platelet production and function

10¹¹ blood platelets are produced daily by bone marrow megakaryocytes (MKs) in the human body. MKs develop from hematopoietic stem cells and, as they mature, form the demarcation membrane system in the cytoplasm, which serves as a membrane reservoir for the formation of long cytoplasmic protrusions (proplatelets) that extend through the endothelium into the blood vessels of the bone marrow. There, individual platelets are released from the proplatelets by shear forces of the blood stream. Reorganization of the MK cytoskeleton is critical to platelet production, however, the precise mechanisms involved remain poorly understood. Thus, the overall aim of our work is to better understand the whole process of platelet “birth” with a special focus on the role of the cytoskeleton in order to gain important insights into the mechanisms of platelet production disorders and to improve in vitro platelet production for transfusion purposes. Platelet adhesion, activation and aggregation on an injured vessel wall are essential for hemostasis, but under pathological conditions can lead to thrombotic diseases such as stroke and heart attack. Such thrombotic events are among the leading causes of disability and death worldwide. While the role of platelets in preventing blood loss has been intensively studied with regard to classical signaling cascades, the mechanobiological aspects of thrombus formation are poorly understood. Using genetically-modified mouse lines and blood samples from patients, cell biological assays in combination with microstructured arrays we investigate the importance of platelet cytoskeletal alterations at the level of basic and translational research.

Markus Bender studied Biomedicine at the University of Wuerzburg in Germany. He worked at the Rudolf Virchow Center for Experimental Biomedicine, University and obtained his PhD in 2010. In 2012, he moved with a fellowship funded by the German Research Foundation (DFG) to the Harvard Medical School, Boston, United States. Since 2015, Markus Bender is supported by the Emmy Noether Programme of the DFG as an independent junior research group leader at the Institute of Experimental Biomedicine I, University Hospital Wuerzburg, Germany. Furthermore, he is a principal investigator in the Collaborative Research Center (CRC)/TR240 “Platelets – Molecular, cellular and systemic functions in health and disease”. Very recently, he was accepted to the Heisenberg Programme of the DFG. His research activities are mainly focused on unraveling the mechanisms involved in platelet production, platelet receptor regulation and thrombus formation. He was awarded with the Bayer Thrombosis Research Award 2015 in recognition of his work on the Wiskott-Aldrich syndrome, and received the Alexander Schmidt Award 2020 from the Society for Thrombosis and Hemostasis Research (Gesellschaft für Thrombose- und Hämostaseforschung e.V, GTH) for his outstanding work in the field of hemostaseology.

How growth cone microtubules direct to neuron morphogenesis

Microtubules (MTs) are dynamic cytoskeleton polymers forming a polarized intracellular network that provides structural support, facilitates intracellular transport, and drives chromosome segregation. In developing neurons, MTs that dynamically invade the growth cone of advancing neurites are essential, but the mechanisms by which microtubules guide neuron morphogenesis are incompletely understood. For example, doublecortin (DCX) is a microtubule-associated protein that is frequently mutated in lissencephaly-spectrum neurodevelopmental disorders in which immature neurons fail to migrate correctly through the developing cortex. To better understand how DCX controls microtubule function in developing cortical neurons we generated a human induced pluripotent stem cell (hiPSC) line in which DCX is tagged with eGFP at the endogenous locus by CRISPR/Cas9 genome editing. DCX-eGFP dynamics at physiological expression levels confirm our previous findings that DCX specifically decorates straight GDP-lattice microtubules (Ettinger et al., Curr. Biol. 26:1549-1555). I will discuss our ongoing work in developing neurons asking how doublecortin (DCX) and other microtubule-associated proteins control growth cone MTs and direct growth cone advance and guidance. I will specifically focus on how we use extracellular matrix patterning and microfabrication in ongoing experiments.

Torsten Wittmann is a professor in the Department of Cell and Tissue Biology at the University of California, San Francisco. Research in the Wittmann lab focusses on how the microtubule cytoskeleton organizes intracellular structure and controls complex cell dynamics such as migration, polarity, and division that are critical to normal and pathological cell biology. Prof. Wittmann received his PhD from the University of Heidelberg / European Molecular Biology Laboratory in 1999 and since then has worked on many aspects of intracellular dynamics utilizing advanced live cell microscopy and optogenetics approaches.