scholarly journals Charge-controlled microfluidic formation of lipid-based single- and multicompartment systems

Lab on a Chip ◽  
2018 ◽  
Vol 18 (17) ◽  
pp. 2665-2674 ◽  
Author(s):  
Barbara Haller ◽  
Kerstin Göpfrich ◽  
Martin Schröter ◽  
Jan-Willi Janiesch ◽  
Ilia Platzman ◽  
...  
Keyword(s):  
High Throughput ◽  
Cell Model ◽  
Model Systems ◽  
Giant Unilamellar Vesicles ◽  
Unilamellar Vesicles ◽  
On Demand ◽  
Demand Creation ◽  
Synthetic Cell ◽  
Multicompartment Systems

We introduce a high-throughput microfluidic off-the-shelf approach for the on-demand creation of giant unilamellar vesicles (GUVs) or multicompartment synthetic cell model systems.

scholarly journals Atomic force microscopy of phase separation on ruptured, giant unilamellar vesicles

2018 ◽  
Author(s):  
Yanfei Jiang ◽  
Guy M. Genin ◽  
Kenneth M. Pryse ◽  
Elliot L. Elson
Keyword(s):  
Atomic Force Microscopy ◽  
Phase Separation ◽  
Model Systems ◽  
Study Phase ◽  
Dark Phase ◽  
Giant Unilamellar Vesicles ◽  
Unilamellar Vesicles ◽  
Force Microscopy ◽  
Atomic Force ◽  
Lipid Species

AbstractGiant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs) are synthetic model systems widely used in biophysical studies of lipid membranes. Phase separation behaviors of lipid species in these two model systems differ due to the lipid-substrate interactions that are present only for SLBs. Therefore, GUVs are believed to resemble natural cell membranes more closely, and a very large body of literature focuses on applying nano-characterization techniques to quantify phase separation on GUVs. However, one important technique, atomic force microscopy (AFM), has not yet been used successfully to study phase separation on GUVs. In the present study, we report that in binary systems, certain phase domains on GUVs retain their original shapes and patterns after the GUVs rupture on glass surfaces. This enabled AFM experiments on phase domains from binary GUVs containing 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). These DLPC/DSPC and DLPC/DPPC GUVs both presented two different gel phases, one of which (bright phase) included a relatively high concentration of DiI-C20 but excluded Bodipy-HPC, and the other of which (dark phase) excluded both probes. The bright phases are of interest because they seem to stabilize dark phases against coalescence. Results suggested that the gel phases labeled by DiI-C20 in the DLPC/DSPC membrane, which surround the dark gel phase, is an extra layer of membrane, indicating a highly curved structure that might stabilize the interior dark domains. This phenomenon was not found in the DLPC/DPPC membrane. These results show the utility of AFM on collapsed GUVs, and suggest a possible mechanism for stabilization of lipid domains.

Fluorescent probe partitioning in giant unilamellar vesicles of ‘lipid raft’ mixtures

2010 ◽  
Vol 430 (3) ◽  
pp. 415-423 ◽  
Author(s):  
Janos Juhasz ◽  
James H. Davis ◽  
Frances J. Sharom
Keyword(s):  
Fluorescent Probe ◽  
Lipid Bilayer ◽  
Fluorescent Probes ◽  
Phase Behaviour ◽  
Model Systems ◽  
Fluorescence Probes ◽  
Lipid Phase ◽  
Giant Unilamellar Vesicles ◽  
Unilamellar Vesicles ◽  
Lipid Mixture

Direct visualization of raft-like lo (liquid-ordered) domains in model systems and cells using microscopic techniques requires fluorescence probes with known partitioning preference for one of the phases present. However, fluorescent probes may display dissimilar partitioning preferences in different lipid sys-tems and can also affect the phase behaviour of the host lipid bilayer. Therefore a detailed understanding of the behaviour of fluorescent probes in defined lipid bilayer systems with known phase behaviour is essential before they can be used for identifying domain phase states. Using giant unilamellar vesicles composed of the ternary lipid mixture DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)/DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine)/cholesterol, for which the phase behaviour is known, we examined nine commonly used fluorescent probes using confocal fluorescence microscopy. The partitioning preference of each probe was assigned either on the basis of quantification of the domain area fractions or by using a well-characterized ld (liquid-disordered)-phase marker. Fluorescent probes were examined both individually and using dual or triple labelling approaches. Most of the probes partitioned individually into the ld phase, whereas only NAP (naphtho[2,3-a]pyrene) and NBD-DPPE [1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl] preferred the lo phase. We found that Rh-DPPE (Lissamine™ rhodamine B–1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) increased the miscibility transition temperature, Tmix. Interestingly, the partitioning of DiIC18 (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) was influenced by Bodipy®-PC [2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexa-decanoyl-sn-glycero-3-phosphocholine]. The specific use of each of the fluorescent probes is determined by its photostability, partitioning preference, ability to detect lipid phase separations and induced change in Tmix. We demonstrate the importance of testing a specific fluorescent probe in a given model membrane system, rather than assuming that it labels a particular lipid phase.

scholarly journals Surfactant-free production of biomimetic giant unilamellar vesicles using PDMS-based microfluidics

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Naresh Yandrapalli ◽  
Julien Petit ◽  
Oliver Bäumchen ◽  
Tom Robinson
Keyword(s):  
High Throughput ◽  
Paradigm Shift ◽  
Mammalian Cells ◽  
Lipid Vesicles ◽  
Giant Unilamellar Vesicles ◽  
Unilamellar Vesicles ◽  
Artificial Cells ◽  
Membrane Function ◽  
Lipid Diffusion ◽  
Protein Incorporation

AbstractMicrofluidic production of giant lipid vesicles presents a paradigm-shift in the development of artificial cells. While production is high-throughput and the lipid vesicles are mono-disperse compared to bulk methods, current technologies rely heavily on the addition of additives such as surfactants, glycerol and even ethanol. Here we present a microfluidic method for producing biomimetic surfactant-free and additive-free giant unilamellar vesicles. The versatile design allows for the production of vesicle sizes ranging anywhere from ~10 to 130 µm with either neutral or charged lipids, and in physiological buffer conditions. Purity, functionality, and stability of the membranes are validated by lipid diffusion, protein incorporation, and leakage assays. Usability as artificial cells is demonstrated by increasing their complexity, i.e., by encapsulating plasmids, smaller liposomes, mammalian cells, and microspheres. This robust method capable of creating truly biomimetic artificial cells in high-throughput will prove valuable for bottom-up synthetic biology and the understanding of membrane function.

scholarly journals Giant unilamellar vesicles - a perfect tool to visualize phase separation and lipid rafts in model systems.

2009 ◽  
Vol 56 (1) ◽  
Author(s):  
Olga Wesołowska ◽  
Krystyna Michalak ◽  
Jadwiga Maniewska ◽  
Andrzej B Hendrich
Keyword(s):  
Phase Separation ◽  
Lipid Membranes ◽  
Average Diameter ◽  
Model Systems ◽  
Membrane Properties ◽  
Lipid Phase ◽  
Giant Unilamellar Vesicles ◽  
Unilamellar Vesicles ◽  
Simple Method ◽  
Wide Range

Model systems such as black lipid membranes or conventional uni- or multilamellar liposomes are commonly used to study membrane properties and structure. However, the construction and dimensions of these models excluded their direct optical microscopic observation. Since the introduction of the simple method of liposome electroformation in alternating electric field giant unilamellar vesicles (GUVs) have become an important model imitating biological membranes. Due to the average diameter of GUVs reaching up to 100 microm, they can be easily observed under a fluorescent or confocal microscope provided that the appropriate fluorescent probe was incorporated into the lipid phase during vesicle formation. GUVs can be formed from different lipid mixtures and they are stable in a wide range of physical conditions such as pH, pressure or temperature. This mini-review presents information about the methods of GUV production and their usage. Particularly, the use of GUVs in studying lipid phase separation and the appearance and behavior of lipid domains (rafts) in membranes is discussed but also other examples of GUVs use in membrane research are given. The experience of the authors in setting up the GUV-forming equipment and production of GUVs is also presented.

scholarly journals Integrating microfluidic generation, handling and analysis of biomimetic giant unilamellar vesicles

Lab on a Chip ◽  
2014 ◽  
Vol 14 (11) ◽  
pp. 1806-1810 ◽  
Author(s):  
D. J. Paterson ◽  
J. Reboud ◽  
R. Wilson ◽  
M. Tassieri ◽  
J. M. Cooper
Keyword(s):  
Antimicrobial Peptides ◽  
High Throughput ◽  
Giant Unilamellar Vesicles ◽  
Unilamellar Vesicles

Integrated microfluidic manufacture and analysis of biomimetic vesicles, demonstrated by a high-throughput dye leakage experiment, using antimicrobial peptides.

scholarly journals Studying the effects of asymmetry on the bending rigidity of lipid membranes formed by microfluidics

2016 ◽  
Vol 52 (30) ◽  
pp. 5277-5280 ◽  
Author(s):  
K. Karamdad ◽  
R. V. Law ◽  
J. M. Seddon ◽  
N. J. Brooks ◽  
O. Ces
Keyword(s):  
Mechanical Properties ◽  
High Throughput ◽  
Lipid Membranes ◽  
Bending Rigidity ◽  
Giant Unilamellar Vesicles ◽  
Unilamellar Vesicles ◽  
Microfluidic Platform

In this article we detail a robust high-throughput microfluidic platform capable of fabricating either symmetric or asymmetric giant unilamellar vesicles (GUVs) and characterise the mechanical properties of their membranes.

scholarly journals The TGFβ Family in Human Placental Development at the Fetal-Maternal Interface

Biomolecules ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 453
Author(s):  
Susana M. Chuva de Sousa Lopes ◽  
Marta S. Alexdottir ◽  
Gudrun Valdimarsdottir
Keyword(s):  
Stem Cell ◽  
Transforming Growth Factor Beta ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Cell Model ◽  
Embryonic Stem ◽  
Model Systems ◽  
Special Focus ◽  
Placental Development

Emerging data suggest that a trophoblast stem cell (TSC) population exists in the early human placenta. However, in vitro stem cell culture models are still in development and it remains under debate how well they reflect primary trophoblast (TB) cells. The absence of robust protocols to generate TSCs from humans has resulted in limited knowledge of the molecular mechanisms that regulate human placental development and TB lineage specification when compared to other human embryonic stem cells (hESCs). As placentation in mouse and human differ considerably, it is only with the development of human-based disease models using TSCs that we will be able to understand the various diseases caused by abnormal placentation in humans, such as preeclampsia. In this review, we summarize the knowledge on normal human placental development, the placental disease preeclampsia, and current stem cell model systems used to mimic TB differentiation. A special focus is given to the transforming growth factor-beta (TGFβ) family as it has been shown that the TGFβ family has an important role in human placental development and disease.

scholarly journals Effect of the Membrane Composition of Giant Unilamellar Vesicles on Their Budding Probability: A Trade-Off between Elasticity and Preferred Area Difference

Life ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 634
Author(s):  
Ylenia Miele ◽  
Gábor Holló ◽  
István Lagzi ◽  
Federico Rossi
Keyword(s):  
Phospholipid Fatty Acid ◽  
Enzymatic Reaction ◽  
Stimuli Responsive ◽  
Membrane Composition ◽  
Giant Unilamellar Vesicles ◽  
Physical Processes ◽  
Unilamellar Vesicles ◽  
Division Process ◽  
The Origin Of Life ◽  
Area Difference

The budding and division of artificial cells engineered from vesicles and droplets have gained much attention in the past few decades due to an increased interest in designing stimuli-responsive synthetic systems. Proper control of the division process is one of the main challenges in the field of synthetic biology and, especially in the context of the origin of life studies, it would be helpful to look for the simplest chemical and physical processes likely at play in prebiotic conditions. Here we show that pH-sensitive giant unilamellar vesicles composed of mixed phospholipid/fatty acid membranes undergo a budding process, internally fuelled by the urea–urease enzymatic reaction, only for a given range of the membrane composition. A gentle interplay between the effects of the membrane composition on the elasticity and the preferred area difference of the bilayer is responsible for the existence of a narrow range of membrane composition yielding a high probability for budding of the vesicles.

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