We present here a microfluidic device that generates sub-millimetric hollow hydrogel spheres, encapsulating cells and coated internally with a layer of reconstituted extracellular matrix (ECM) of a few microns thick. The spherical capsules, composed of alginate hydrogel, originate from the spontaneous instability of a multi-layered jet formed by co-extrusion using a coaxial flow device. We provide a simple design to manufacture this device using a DLP (digital light processing) 3D printer. Then, we demonstrate how the inner wall of the capsules can be decorated with a continuous ECM layer that is anchored to the alginate gel and mimics the basal membrane of a cellular niche. Finally, we used this approach to encapsulate human Neural Stem Cells (hNSC) derived from human Induced Pluripotent Stem Cells (hIPSC), which were further differentiated into neurons within the capsules with negligible loss of viability. Altogether, we show that these capsules may serve as cell micro-containers compatible with complex cell culture conditions and applications. These developments widen the field of research and biomedical applications of the cell encapsulation technology.
Highlights d A proliferating epithelium encapsulated in a hollow sphere spontaneously invaginates d Epithelial proliferation generates compressive stresses that deform the elastic shell d Coupling between stress and folding shape shows that folding arises from buckling d Epithelial elastic moduli are inferred from buckling theory and experiments
The generation of pulling and pushing forces is one of the important functions of microtubules, which are dynamic and polarized structures. The ends of dynamic microtubules are able to form relatively stable links to cellular structures, so that when a microtubule grows it can exert a pushing force and when it shrinks it can exert a pulling force. Microtubule growth and shrinkage are tightly regulated by microtubule-associated proteins (MAPs) that bind to microtubule ends. Given their localization, MAPs may be exposed to compressive and tensile forces. The effect of such forces on MAP function, however, is poorly understood. Here we show that beads coated with the microtubule polymerizing protein XMAP215, the Xenopus homolog of Dis1 and chTOG, are able to link stably to the plus ends of microtubules, even when the ends are growing or shrinking; at growing ends, the beads increase the polymerization rate. Using optical tweezers, we found that tensile force further increased the microtubule polymerization rate. These results show that physical forces can regulate the activity of MAPs. Furthermore, our results show that XMAP215 can be used as a handle to sense and mechanically manipulate the dynamics of the microtubule tip.microtubule polymerase | microtubule dynamics | optical trap X MAP215 is a microtubule plus-end binding protein that alters microtubule dynamics (1-4) by promoting microtubule growth. It was identified as a factor increasing microtubule polymerization rates in Xenopus egg extracts (5). In vivo studies have shown that disruption of XMAP215 function leads to short interphase microtubules, reduced microtubule growth rate, and increased frequency of microtubule catastrophe and pause events (6-11). Depletion of XMAP215 also results in short, abnormally formed mitotic spindles and short astral microtubules (7,12,13). In vitro studies have demonstrated that XMAP215 binds preferentially to the microtubule plus end and follows the growth and shrinkage of the microtubule tip, catalyzing microtubule growth (14-16). In addition to localization at the microtubule plus end and centrosomes (17-19), proteins of the XMAP215/Dis1 family are found at the kinetochores (9, 20, 21), which are sites of high force during cell division. Microtubules themselves can exert pulling and pushing forces (22)(23)(24)(25)(26)(27)(28)(29)(30). Therefore, the localization of XMAP215 at microtubule ends suggests that XMAP215 can function under load. However, the effect of forces on XMAP215 activity has not been directly analyzed. In this study, we investigated the influence of forces-directed toward the microtubule plus and minus end-on XMAP215 activity in vitro. Microtubule growth and shrinkage were monitored with high spatial and temporal resolution by visualizing the position of XMAP215-coated polystyrene microspheres bound to the tips of dynamic microtubules. The microspheres, so-called "beads," served as handles to apply forces on XMAP215 molecules using optical tweezers. Results XMAP215-Coated Beads Remain Attached to Growing ...
Many organs, such as the gut or the spine are formed through folding of an epithelium. Whereas genetic regulation of epithelium folding has been investigated extensively, the nature of the mechanical forces driving this process remain largely unknown. Here we show that monolayers of identical cells proliferating on the inner surface of elastic spherical shells can spontaneously fold. By measuring the elastic deformation of the shell we inferred the forces acting within the monolayer. Using analytical and numerical theories at different scales, we found that the compressive stresses arising within the cell monolayer through proliferation quantitatively account for the shape of folds observed in experiments. Our study shows that forces arising from epithelium growth are sufficient to drive folding by buckling.
Microspheres are often used as handles for protein purification or force spectroscopy. For example, optical tweezers apply forces on trapped particles to which motor proteins are attached. However, even though many attachment strategies exist, procedures are often limited to a particular biomolecule and prone to non-specific protein or surface attachment. Such interactions may lead to loss of protein functionality or microsphere clustering. Here, we describe a versatile coupling procedure for GFP-tagged proteins via a polyethylene glycol linker preserving the functionality of the coupled proteins. The procedure combines well-established protocols, is highly reproducible, reliable, and can be used for a large variety of proteins. The coupling is efficient and can be tuned to the desired microsphere-to-protein ratio. Moreover, microspheres hardly cluster or adhere to surfaces. Furthermore, the procedure can be adapted to different tags providing flexibility and a promising attachment strategy for any tagged protein.
Results of investigation of the elastic modulus for cartilage tissue using a technique of micro- and nanoindentation performed with help of an atomic force microscope are presented. SEM and AFM methods were applied to visualize a topography of surface layers of the entire cartilage and as well as its slices and thus to reveal features of the collagen fibers orientation. The technique used for a quantitative evaluation of the elastic modulus under compression against a ball microindenter (curvature radius - 350 micron) and a nanoindenter (30 nm) is described. It was shown that the cartilage behavior is highly stabile under the load if the entire composite structure of cartilage tissue is engaged into the deformation process. Tribological characteristics were investigated using the ball indenter oscillated by a tuning fork. Dependence of the friction coefficient from applied loads was obtained that revealed strong influence of an interstitial fluid on friction properties. Friction coefficient of a rat cartilage tissue as 0.08 was obtained using a developed plant prototype for tribological measurements based on the AFM construction.
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