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Tubular membrane structures are widespread in eukaryotic cells, but the mechanisms underlying their formation and stability are not well understood. Previous work has focused on tube extrusion from cells and model membranes under the application of external forces. Here, we present novel membrane/polymer systems, where stable tubes form in the absence of externally applied forces. Solutions of two water-soluble polymers, polyethylene glycol and dextran, were encapsulated in giant lipid vesicles, cell-size model systems. Hypertonic deflation induced phase separation of the enclosed solution. The excess membrane area created during the deflation process was stored in a large number of membrane nanotubes inside the vesicle. The tubes had a diameter below optical resolution and became visible only when fluorescently labeled. The tubes were rather stable: In the absence of external forces, they existed for several days. A theoretical analysis of the shapes of the deflated vesicles reveals that these shapes would be unstable if the membranes had no spontaneous curvature. Using the large separation of length scales between the tube diameter and the overall size of the vesicles, the spontaneous curvature can be calculated and is found to be about -1/(240 nm) for a certain range of polymer concentrations. The nanotubes could also be retracted back into the mother vesicle by increasing the membrane tension via micropipette aspiration of the vesicle. Membrane tubes, which can form and be retracted easily, should be relevant for lipid storage in cells.

Giant vesicles are widely used as a model membrane system, both for basic biological systems and for their promising applications in the development of smart materials and cell mimetics, as well as in driving new technologies in synthetic biology and for the cosmetics and pharmaceutical industry. The reader is guided to use giant vesicles, from the formation of simple membrane platforms to advanced membrane and cell system models. It also includes fundamentals for understanding lipid or polymer membrane structure, properties and behavior. Every chapter includes ideas for further applications and discussions on the implications of the observed phenomena towards understanding membrane-related processes. The Giant Vesicle Book is meant to be a road companion, a trusted guide for those making their first steps in this field as well as a source of information required by experts. Key Features • A complete summary of the field, covering fundamental concepts, practical methods, core theory, and the most promising applications • A start-up package of theoretical and experimental information for newcomers in the field • Extensive protocols for establishing the required preparations and assays • Tips and instructions for carefully performing and interpreting measurements with giant vesicles or for observing them, including pitfalls • Approaches developed for investigating giant vesicles as well as brief overviews of previous studies implementing the described techniques • Handy tables with data and structures for ready reference Part I: The making of Chapter 1 Preparation methods for giant unilamellar vesicles - Rumiana Dimova, Pasquale Stano, Carlos M. Marques and Peter Walde Chapter 2 Preparation and properties of giant plasma membrane vesicles and giant unilamellar vesicles from natural membranes - Joseph H. Lorent and Ilya Levental Chapter 3 Protein reconstitution in giant vesicles - Matthias Garten, Daniel Lévy and Patricia Bassereau　 Chapter 4 GUVs with cytoskeleton - Tobias Härtel and Petra Schwille Part II: Giant vesicles theoretically and in silico Chapter 5 Understanding giant vesicles – a theoretical perspective - Reinhard Lipowsky Chapter 6 Simulating membranes, vesicles, and cells - Thorsten Auth, Dmitry A. Fedosov and Gerhard Gompper Chapter 7 Theory of vesicle dynamics in flow and electric fields - Petia M. Vlahovska and Chaouqi Misbah Chapter 8 Particle-membrane interactions - Jaime Agudo-Canalejo, Reinhard Lipowsky Chapter 9 Theory of polymer-membrane interactions - Fabrice Thalmann and Carlos M. Marques Part III: GUV-based techniques and what one can learn from them Chapter 10 Application of optical microscopy techniques on giant unilamellar vesicles - Luis A. Bagatolli Chapter 11 Mechanics assays of synthetic lipid membranes based on micropipette aspiration - Elisa Parra and David Needham Chapter 12 Atomic force microscopy of giant unilamellar vesicles - Andreas Janshoff Chapter 13 Manipulation and biophysical characterization of GUVs with an optical stretcher - Gheorghe Cojoc, Antoine Girot, Ulysse Delabre and Jochen Guck Chapter 14 Vesicle fluctuation analysis - John Hjort Ipsen, Allan Grønhøj Hansen and Tripta Bhatia Chapter 15 Using electric fields to assess membrane material properties in GUVs - Rumiana Dimova and　　Karin A. Riske Chapter 16 Creating membrane nanotubes from GUVs - Coline Prévost, Mijo Simunovic and Patricia Bassereau Chapter 17 Measuring GUV adhesion - Kheya Sengupta and Ana Smith Chapter 18 Phase diagrams and tie lines in GUVs - Matthew C. Blosser, Caitlin Cornell, Scott P. Rayermann and Sarah L. Keller Chapter 19 Vesicle dynamics in flow: an experimental approach - Victor Steinberg and Michael Levant Chapter 20 Membrane permeability measurements - Begoña Ugarte-Uribe, Ana J. García-Sáez and Mireille M. A. E. Claessens Part IV: GUVs as membrane interaction platforms Chapter 21 - Lipid and protein mobility in GUVs - Begoña Ugarte-Uribe, Kushal Kumar Das and Ana J. García-Sáez Chapter 22 Shining light on membranes - Rosangela Itri, Carlos M. Marques and Mauricio S. Baptista Chapter 23 Protein-membrane interactions - Eva M Schmid and Daniel A Fletcher Chapter 24 Effects of antimicrobial peptides and detergents on GUVs - Karin A. Riske Chapter 25 Lipid-polymer interactions: effect on GUVs shapes and behavior - Brigitte Pépin-Donat, François Quemeneur and Clément Campillo Part V: GUVs as complex membrane containers Chapter 26 Polymersomes - Praful Nair, David Christian and Dennis E. Discher Chapter 27 Giant hybrid polymer/lipid vesicles - Thi Phuong Tuyen Dao, Khalid Ferji, Fabio Fernandes, Manuel Prieto, Sébastien Lecommandoux, Emmanuel Ibarboure, Olivier Sandre and Jean-François Le Meins Chapter 28 Giant unilamellar vesicles: from protocell models to the construction of minimal cells - Masayuki Imai and Peter Walde Chapter 29 Encapsulation of aqueous two-phase systems and gels within giant lipid vesicles - Allyson M. Marianelli and Christine D. Keating Chapter 30 Droplet-supported giant lipid vesicles as compartments for synthetic biology - Johannes P. Frohnmayer, Marian Weiss, Lucia T. Benk, Jan-Willi Janiesch, Barbara Haller, Rafael B. Lira, Rumiana Dimova, Ilia Plazman and Joachim P. Spatz Appendices Appendix 1 List of lipids and physical constants of lipid bilayers Appendix 2 List of membrane dyes and fluorescent groups conjugated to lipids Appendix 3 List of detergents Appendix 4 List of water-soluble dyes or their fluorescent groups and their structures Rumiana Dimova leads an experimental lab in biophysics at the Max Planck Institute of Colloids and Interfaces in Potsdam, Germany. She has been working with giant vesicles already from the beginning of her scientific career. After being introduced into the magic of their preparation during her studies as a student in Bulgaria, she remained fascinated by their application and over the years pursued a variety of projects employing giant vesicles as a platform to develop new methods for the biophysical characterization of membranes and processes involving them. Until now, these studies have resulted in more than hundred peer-reviewed publications. Recently, she was also awarded the Emmy Noether distinction for women in physics of the European Physical Society. Carlos Marques , a CNRS senior scientist, founded the MCube group at the Charles Sadron Institute in Strasbourg, France, where he gears experimental and theoretical research towards the understanding of the physical properties of self-assembled lipid bilayers. Trained as a polymer theoretician, Carlos first got interested in membranes because they interact with polymers and published the first prediction for the membrane changes expected when polymers adsorb on lipid bilayers. He then expanded the scope of his group to include experiments and numerical simulations, and has now published many papers based on research with giant unilamellar vesicles, including the first study of lipid oxidation in GUVs and the discovery of the so-called PVA method for vesicle growth.

With the increasing concern of neurodegenerative diseases, the development of new therapies and effective pharmaceuticals targeted to central nervous system (CNS) illnesses is crucial for ensuring social and economic sustainability in an ageing world. Unfortunately, many promising treatments at the initial stages of the pharmaceutical development process, that is at the in vitro screening stages, do not finally show the expected results at the clinical level due to their inability to cross the human blood-brain barrier (BBB), highlighting the inefficiency of in vitro BBB models to recapitulate the real functionality of the human BBB. In the last decades research has focused on the development of in vitro BBB models from basic 2D monolayer cultures to 3D cell co-cultures employing different system configurations. Particularly, the use of polymeric hollow fiber membranes (HFs) as scaffolds plays a key role in perfusing 3D dynamic in vitro BBB (DIV-BBB) models. Their incorporation into a perfusion bioreactor system may potentially enhance the vascularization and oxygenation of 3D cell cultures improving cell communication and the exchange of nutrients and metabolites through the microporous membranes. The quest for developing a benchmark 3D dynamic in vitro blood brain barrier model requires the critical assessment of the different aspects that limits the technology. This article will focus on identifying the advantages and main limitations of the HFs in terms of polymer materials, microscopic porous morphology, and other practical issues that play an important role to adequately mimic the physiological environment and recapitulate BBB architecture. Based on this study, we consider that future strategic advances of this technology to become fully implemented as a gold standard DIV-BBB model will require the exploration of novel polymers and/or composite materials, and the optimization of the morphology of the membranes towards thinner HFs (<50 μm) with higher porosities and surface pore sizes of 1–2 µm to facilitate the intercommunication via regulatory factors between the cell co-culture models of the BBB.

In the present study, cellulose, one of the most abundant natural polymers, was adopted as a membrane material for optical resolution. The membrane was prepared from cellulose, and it selectively transported l -glutamic acid ( l -Glu) from racemic mixtures of Glu with a concentration gradient as the driving force. A molecularly imprinted cellulose membrane, which was constructed from cellulose and l -Glu, showed d -glutamic acid ( d -Glu) permselectivity, which is thought to be due to a strong interaction between l -Glu and the molecularly imprinted cellulose membrane. The performance of both membranes was significantly dependent on LiCl, which was used to dissolve cellulose into an N , N -dimethylactamide (DMAc) solvent. The permselectivity increased with the LiCl concentration and asymptotically reached a maximum value at higher LiCl concentrations. The affinity constant of LiCl for the LiCl recognition sites in both membranes was 65 mol −1 dm 3 . Applications of l -Glu molecularly imprinted cellulose membranes to chiral separation were studied. LiCl, which was adopted to dissolve cellulose into solvent, N , N -dimethylactamide (DMAc), worked well as a print molecule to construct recognition sites toward Li + (or LiCl). The chiral separation ability was greatly dependent on LiCl. The permselectivity was increased with the LiCl concentration and asymptotically reached the maximum value at higher LiCl concentration region. The affinity constants between LiCl and LiCl recognition sites in both control and l -Glu molecularly imprinted membranes were determined to be 65 mol −1 dm 3 .