Stem cells sense and respond to the mechanical properties of the extracellular matrix. However, both the extent to which extracellular matrix mechanics affect stem cell fate in 3D micro-environments and the underlying biophysical mechanisms are unclear. We demonstrate that the commitment of mesenchymal stem cell (MSC) populations changes in response to the rigidity of 3D micro-environments, with osteogenesis occurring predominantly at 11–30 kPa. In contrast to previous 2D work, however, cell fate was not correlated with morphology. Instead, matrix stiffness regulated integrin binding as well as reorganization of adhesion ligands on the nanoscale, both of which were traction-dependent and correlated with osteogenic commitment of MSC populations. These findings suggest that cells interpret changes in the physical properties of adhesion substrates as changes in adhesion ligand presentation, and that cells themselves can be harnessed as tools to mechanically process materials into structures that feedback to manipulate their fate.

Cells alter their mechanical properties in response to their local microenvironment; this plays a role in determining cell function and can even influence stem cell fate. Here, we identify a robust and unified relationship between cell stiffness and cell volume. As a cell spreads on a substrate, its volume decreases, while its stiffness concomitantly increases. We find that both cortical and cytoplasmic cell stiffness scale with volume for numerous perturbations, including varying substrate stiffness, cell spread area, and external osmotic pressure. The reduction of cell volume is a result of water efflux, which leads to a corresponding increase in intracellular molecular crowding. Furthermore, we find that changes in cell volume, and hence stiffness, alter stem-cell differentiation, regardless of the method by which these are induced. These observations reveal a surprising, previously unidentified relationship between cell stiffness and cell volume that strongly influences cell biology.cell volume | cell mechanics | molecular crowding | gene expression | stem cell fate C ell volume is a highly regulated property that affects myriad functions (1, 2). It changes over the course of the cell life cycle, increasing as the cell plasma membrane grows and the amount of protein, DNA, and other intracellular material increases (3). However, it can also change on a much more rapid time scale, as, for example, on cell migration through confined spaces (4, 5); in this case, the volume change is a result of water transport out of the cell. This causes increased concentration of intracellular material and molecular crowding, having numerous important consequences (6, 7). Alternately, the volume of a cell can be directly changed through application of an external osmotic pressure. This forces water out of the cell, which also decreases cell volume, increases the concentration of intracellular material, and intensifies molecular crowding. Application of an external osmotic pressure to reduce cell volume also has other pronounced manifestations: For example, it leads to a significant change in cell mechanics, resulting in an increase in stiffness (8); it also impacts folding and transport of proteins (9), as well as condensation of chromatin (10). These dramatic effects of osmotic-induced volume change on cell behavior raise the question of whether cells ever change their volume through water efflux under isotonic conditions, perhaps to modulate their mechanics and behavior through changes in molecular crowding.Here, we demonstrate that when cells are cultured under the same isotonic conditions, but under stiffer extracellular environments, they reduce their cell volume through water efflux out of the cell, and this has a large and significant impact on cell mechanics and cell physiology. Specifically, as a cell spreads out on a stiff substrate, its volume decreases, and the cell behaves in a similar manner to that observed for cells under external osmotic pressure: Both the cortical and cytoplasmic stiffness increase as the vol...

Organ and tissue loss through disease and injury motivate the development of therapies that can regenerate tissues and decrease reliance on transplantations. Regenerative medicine, an interdisciplinary field that applies engineering and life science principles to promote regeneration, can potentially restore diseased and injured tissues and whole organs. Since the inception of the field several decades ago, a number of regenerative medicine therapies, including those designed for wound healing and orthopedics applications, have received Food and Drug Administration (FDA) approval and are now commercially available. These therapies and other regenerative medicine approaches currently being studied in preclinical and clinical settings will be covered in this review. Specifically, developments in fabricating sophisticated grafts and tissue mimics and technologies for integrating grafts with host vasculature will be discussed. Enhancing the intrinsic regenerative capacity of the host by altering its environment, whether with cell injections or immune modulation, will be addressed, as well as methods for exploiting recently developed cell sources. Finally, we propose directions for current and future regenerative medicine therapies.regenerative medicine | tissue engineering | biomaterials | review

Existing techniques to encapsulate cells into microscale hydrogels generally yield high polymer-to-cell ratios and lack control over the hydrogel’s mechanical properties1. Here, we report a microfluidic-based method for encapsulating single cells in a ~6 micron layer of alginate that increases the proportion of cell-containing microgels by 10-fold, with encapsulation efficiencies over 90%. We show that in vitro cell viability was maintained over a three-day period, that the microgels are mechanically tractable, and that for microscale cell assemblages of encapsulated marrow stromal cells cultured in microwells, osteogenic differentiation of encapsulated cells depends on gel stiffness and cell density. We also show that intravenous injection of singly-encapsulated marrow stromal cells into mice delays clearance kinetics and sustains donor-derived soluble factors in vivo. The encapsulation of single cells in tunable hydrogels should find use in a variety of tissue engineering and regenerative medicine applications.

Recent studies have shown that basic cellular behavior varies significantly between two- and three-dimensional culture systems. To identify the origins of these fundamental differences the design of reliable and precisely controlled environments is essential. While 2D cell culture is a well-established technique, the fabrication of defined three-dimensional culture models is still challenging. We present a new method for the microfluidic generation of a micron-sized three-dimensional cell culture system. We use a triggered ionic crosslink formation to generate highly monodisperse and structurally homogeneous alginate microbeads. Aqueous droplets containing a mixture of alginate and a water-soluble calcium-EDTA complex are formed by droplet-based microfluidics. In their complexed form, the calcium ions are homogenously distributed inside the droplet but not accessible for the crosslinking process. Acid addition is used to trigger the degradation of the complex, releasing calcium ions on demand that can physically crosslink the alginate chains. A homogeneous hydrogel network is thus generated which can be transferred into an aqueous environment without losing its structural integrity. Single cells can be encapsulated into these controlled microenvironments which provide structural support while allowing for continuous nutrient supply. We encapsulate individual mesenchymal stem cells (MSCs) into the microbeads which show the aspired cell growth and proliferation.

The presented microrobotic platform combines together the advantages of self-folding NIR light sensitive polymer bilayers, magnetic alginate microbeads, and a 3D manipulation system, to propose a solution for targeted, on-demand drug and cell delivery. First feasibility studies are presented together with the potential of the full design.

Stimuli-responsive materials activated by biological signals play an increasingly important role in biotechnology applications. We exploit the programmability of CRISPR-associated nucleases to actuate hydrogels containing DNA as a structural element or as an anchor for pendant groups. After activation by guide RNA–defined inputs, Cas12a cleaves DNA in the gels, thereby converting biological information into changes in material properties. We report four applications: (i) branched poly(ethylene glycol) hydrogels releasing DNA-anchored compounds, (ii) degradable polyacrylamide-DNA hydrogels encapsulating nanoparticles and live cells, (iii) conductive carbon-black–DNA hydrogels acting as degradable electrical fuses, and (iv) a polyacrylamide-DNA hydrogel operating as a fluidic valve with an electrical readout for remote signaling. These materials allow for a range of in vitro applications in tissue engineering, bioelectronics, and diagnostics.

The COVID-19 pandemic highlights the need for diagnostics that can be rapidly adapted and deployed in a variety of settings. Several SARS-CoV-2 variants have shown worrisome effects on vaccine and treatment efficacy, but no current point-of-care (POC) testing modality allows their specific identification. We have developed miSHERLOCK, a low-cost, CRISPR-based POC diagnostic platform that takes unprocessed patient saliva; extracts, purifies, and concentrates viral RNA; performs amplification and detection reactions; and provides fluorescent visual output with only three user actions and 1 hour from sample input to answer out. miSHERLOCK achieves highly sensitive multiplexed detection of SARS-CoV-2 and mutations associated with variants B.1.1.7, B.1.351, and P.1. Our modular system enables easy exchange of assays to address diverse user needs and can be rapidly reconfigured to detect different viruses and variants of concern. An adjunctive smartphone application enables output quantification, automated interpretation, and the possibility of remote, distributed result reporting.