The key roles played by phospholipids in many cellular processes, has led to the development of model systems, to explore both lipid-lipid and lipid-peptide interactions. Biomimetic giant unilamellar ...vesicles represent close facsimiles of in vivo cellular membranes, although currently their widespread use in research is hindered by difficulties involving their integration into high-throughput techniques, for exploring membrane biology intensively in situ. This paper presents an integrated microfluidic device for the production, manipulation and high-throughput analysis of giant unilamellar vesicles. Its utility is demonstrated by exploring the lipid interaction dynamics of the pore-forming antimicrobial peptide melittin, assessed through the release of fluorescent dyes from within biomimetic vesicles, with membrane compositions similar to mammalian plasma membranes.
Current materials used for in vitro 3D cell culture are often limited by their poor similarity to human tissue, batch-to-batch variability and complexity of composition and manufacture. Here, we ...present a “blank slate” culture environment based on a self-assembling peptide gel free from matrix motifs. The gel can be customised by incorporating matrix components selected to match the target tissue, with independent control of mechanical properties. Therefore the matrix components are restricted to those specifically added, or those synthesised by encapsulated cells. The flexible 3D culture platform provides full control over biochemical and physical properties, allowing the impact of biochemical composition and tissue mechanics to be separately evaluated in vitro. Here, we demonstrate that the peptide gels support the growth of a range of cells including human induced pluripotent stem cells and human cancer cell lines. Furthermore, we present proof-of-concept that the peptide gels can be used to build disease-relevant models. Controlling the peptide gelator concentration allows peptide gel stiffness to be matched to normal breast (<1 kPa) or breast tumour tissue (>1 kPa), with higher stiffness favouring the viability of breast cancer cells over normal breast cells. In parallel, the peptide gels may be modified with matrix components relevant to human breast, such as collagen I and hyaluronan. The choice and concentration of these additions affect the size, shape and organisation of breast epithelial cell structures formed in co-culture with fibroblasts. This system therefore provides a means of unravelling the individual influences of matrix, mechanical properties and cell-cell interactions in cancer and other diseases.
•We propose an optimised self-assembling peptide gel for defined 3D cell culture.•Peptide gels support growth of multiple cell types, including stromal co-culture.•Gels allow independent control of matrix stiffness and ECM functionalisation.•Stiffness, ECM and stromal cells affect growth of breast cancer progression models.•We show proof-of-concept utilising peptide gels to build disease-relevant models.
We demonstrate a new method for measuring the sedimentation of a single colloidal bead by using a combination of optical tweezers and a stereo microscope based on a spatial light modulator. We use ...optical tweezers to raise a micron-sized silica bead to a fixed height and then release it to observe its 3D motion while it sediments under gravity. This experimental procedure provides two independent measurements of bead diameter and a measure of Faxén's correction, where the motion changes due to presence of the boundary.
The viscosity of a fluid can be measured by tracking the motion of a suspended micron-sized particle trapped by optical tweezers. However, when the particle density is high, additional particles ...entering the trap compromise the tracking procedure and degrade the accuracy of the measurement. In this work we introduce an additional Laguerre-Gaussian, i.e. annular, beam surrounding the trap, acting as an optical shield to exclude contaminating particles.
Solutions of intact cardiac thin filaments were examined with transmission electron microscopy, dynamic light scattering (DLS), and particle-tracking microrheology. The filaments self-assembled in ...solution with a bell-shaped distribution of contour lengths that contained a population of filaments of much greater length than the in vivo sarcomere size (∼1
μm) due to a one-dimensional annealing process. Dynamic semiflexible modes were found in DLS measurements at fast timescales (12.5
ns–0.0001
s). The bending modulus of the fibers is found to be in the range 4.5–16
×
10
−27
Jm and is weakly dependent on calcium concentration (with Ca
2+
≥
without Ca
2+). Good quantitative agreement was found for the values of the fiber diameter calculated from transmission electron microscopy and from the initial decay of DLS correlation functions: 9.9
nm and 9.7
nm with and without Ca
2+, respectively. In contrast, at slower timescales and high polymer concentrations, microrheology indicates that the cardiac filaments act as short rods in solution according to the predictions of the Doi-Edwards chopsticks model (viscosity,
η
∼
c
3, where
c is the polymer concentration). This differs from the semiflexible behavior of long synthetic actin filaments at comparable polymer concentrations and timescales (elastic shear modulus,
G′
∼
c
1.4, tightly entangled) and is due to the relative ratio of the contour lengths (∼30). The scaling dependence of the elastic shear modulus on the frequency (
ω) for cardiac thin filaments is
G′
∼
ω
3/4
±
0.03
, which is thought to arise from flexural modes of the filaments.
Microrheology with optical tweezers Yao, Alison; Tassieri, Manlio; Padgett, Miles ...
Lab on a chip,
01/2009, Letnik:
9, Številka:
17
Journal Article
Recenzirano
Microrheology is the study of the flow of materials over small scales. It is of particular interest to those involved with investigations of fluid properties within Lab-on-a-Chip structures or within ...other micron-scale environments. The article briefly reviews existing active and passive methods used in the study of fluids. It then explores in greater detail the use of optical tweezers as an emerging method to investigate rheological phenomena, including, for example, viscosity and viscoelasticity, as well as the related topic of flow. The article also describes, briefly, potential future applications of this topic, in the fields of biological measurement, in general, and Lab-on-a-Chip, in particular.
Photon correlation spectroscopy is used to study the internal dynamics of self-assembled charged peptide fibrils. Short neutral and charged polymeric aggregates have diffusive modes due to whole ...macromolecular motion. For long semiflexible fibrils the logarithm of the intermediate scattering function follows a q 2 t 3/4 scaling at long times consistent with a Kratky−Porod free energy and preaveraged Oseen hydrodynamics. Persistence lengths on the order of micrometers are calculated for the peptide fibrils consistent with estimates from the liquid-crystalline phase behavior. Fibril diameters (5−35 nm) calculated from the initial decay of the correlation functions are in agreement with transmission electron microscopy measurements.
In this work, we present an experimental methodology to investigate the dynamics under shear flow of a drop that is gelling as a consequence of a temperature quench. The experiments were carried out ...on the system water/gellan/
κ-carrageenan in the biphasic region of the phase diagram, the gellan–rich phase being used as the dispersed phase. Gelation was brought about by lowering the temperature during flow after steady state drop deformation had been reached. Simple shear flow was applied by using a parallel plate apparatus equipped with optical microscopy and image analysis, which made it possible to monitor drop shape evolution before, during, and after gelation. The onset of gelation trapped drop deformation, thus producing anisotropic particles. The fingerprint of gelation was the simultaneous tumbling of the drops, which rotated as rigid ellipsoids under the action of shear flow. Interfacial tension between the two equilibrium phases was determined at different times during the temperature quench by analyzing drop retraction upon cessation of flow. Up to gelation, no significant change was observed in the measured values.
The rheological behaviour of suspensions is influenced by many parameters, one of which is the particle shape. For rigid particle suspensions a number of studies demonstrate the effects of the ...particle aspect ratio. Indeed, fibres are widely used as rheology modifiers in different materials such as synthetic polymers. This work is concerned with testing the hypothesis that regularly shaped particles with aspect ratios larger than one that are made of gelled biopolymers could be used as rheology modifiers for biopolymer solutions. Biopolymers, and mixtures thereof are a widely used ingredient in foods and other products with structure functionality. Tailoring rheology modifiers by morphology offers an alternative to using different biopolymers.It is demonstrated how biopolymer suspensions with regular spheroidal, or cylindrical particle shapes can be produced by gelling the droplet phase of a liquid two phase biopolymer mixture in a shear field. Biopolymers were chosen such that gelation is initiated by cooling. Shear-cooling at constant stresses leads to the formation of ellipsoidal particles. Cylindrical particles can be generated by stepping up the shear stress prior to gelation, i.e., stretching the droplet phase into fibrils, and trapping the shape prior to break-up through gelation.Morphologies and steady shear rheological data for suspensions of the two biopolymers gellan and κ-carrageenan with an internal phase volume of 0.2 are reported. The influence of particle shape on relative viscosity is pronounced. At high shear stresses particle orientation leads to decreased viscosity with increasing particle aspect ratio. In the low shear region, higher aspect ratio suspensions show higher viscosities. Additionally, the material properties, including the interfacial tension, which influence the suspension morphology are reported.
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