Dispase/collagenase cocktail allows for coisolation of satellite cells and fibroadipogenic progenitors from human skeletal muscle.

Satellite cells (SCs) and fibroadipogenic progenitors (FAPs) are progenitor populations found in muscle that form new myofibers postinjury. Muscle development, regeneration, and tissue-engineering experiments require robust progenitor populations, yet their isolation and expansion are difficult given their scarcity in muscle, limited muscle biopsy sizes in humans, and lack of methodological detail in the literature. Here, we investigated whether a dispase and collagenase type 1 and 2 cocktail could allow dual isolation of SCs and FAPs, enabling significantly increased yield from human skeletal muscle. Postdissociation, we found that single cells could be sorted into CD56 + CD31-CD45- (SC) and CD56-CD31-CD45- (FAP) cell populations, expanded in culture, and characterized for lineage-specific marker expression and differentiation capacity; we obtained ∼10% SCs and ∼40% FAPs, with yields twofold better than what is reported in current literature. SCs were PAX7+ and retained CD56 expression and myogenic fusion potential after multiple passages, expanding up to 1012 cells. Conversely, FAPs expressed CD140a and differentiated into either fibroblasts or adipocytes upon induction. This study demonstrates robust isolation of both SCs and FAPs from the same muscle sample with SC recovery more than two times higher than previously reported, which could enable translational studies for muscle injuries.NEW & NOTEWORTHY We demonstrated that a dispase/collagenase cocktail allows for simultaneous isolation of SCs and FAPs with 2× higher SC yield compared with other studies. We provide a thorough characterization of SC and FAP in vitro expansion that other studies have not reported. Following our dissociation, SCs and FAPs were able to expand by up to 1012 cells before reaching senescence and maintained differentiation capacity in vitro demonstrating their efficacy for clinical translation for muscle injury.

Mesenchymal stem cell-secretome laden photopolymerizable hydrogels for wound healing.

Mesenchymal stem cell-derived secretome represents an emerging acellular therapeutic which possess significant opportunity for clinical applications due to its anti-inflammatory, immunomodulatory, and wound healing properties. However, maintaining therapeutic efficacy and ensuring stability of cell-based products is challenging, requiring a robust delivery method. Therefore, we designed a hydrogel-based scaffold loaded with CK Cell Technologies' proprietary Mesenchymal stem cell-secretome for controlled release treatment of acute and chronic wounds. We incorporated both conditioned media (CM) and extracellular vesicles (EVs) into gelatin methacryloyl (GelMA) hydrogels and demonstrated how we can tune the diffusive release of the EVs from them. To demonstrate viability of the approach, we developed a wound healing scratch assay where we see in situ release of CM and EVs promote enhanced migration of human dermal fibroblasts (hDFs). We see the colocalization of these EVs in the fibroblasts using fluorescent microscopy. Finally, as a surrogate for in vivo neovascularization, we conducted an in vitro tube formation assay for the MSC-secretome using matrigel-embedded human microvascular endothelial cells. By adding CM and EVs, we observe an increase in tubulogenesis. Collectively, our data demonstrates by tuning the GelMA properties, we can influence the controlled release of the MSC-secretome for a wound dressing and bandage application for chronic and acute wounds.

Using biophysical cues and biomaterials to improve genetic models.

With the advent of induced pluripotent stem cells and modern differentiation protocols, many advances in our understanding of disease have been made possible by in vitro disease modeling; in some cases, their use may have supplanted animal models. Yet in vitro models often rely on rigid cell culture substrates that could limit our ability to completely reproduce human disease in a dish. Nascent work, however, suggests that the combination of biomaterials and/or advanced microphysiological systems-which better recapitulate tissue properties-with stem cells expressing disease mimicking genetics, could substantially improve current disease modeling efforts where genetics alone is insufficient. This review will highlight such recent advances as well as review current challenges that the fields must overcome to create more personalized therapeutics in the future.

Hierarchical assembly of tryptophan zipper peptides into stress-relaxing bioactive hydrogels.

Soft materials in nature are formed through reversible supramolecular assembly of biological polymers into dynamic hierarchical networks. Rational design has led to self-assembling peptides with structural similarities to natural materials. However, recreating the dynamic functional properties inherent to natural systems remains challenging. Here we report the discovery of a short peptide based on the tryptophan zipper (trpzip) motif, that shows multiscale hierarchical ordering that leads to emergent dynamic properties. Trpzip hydrogels are antimicrobial and self-healing, with tunable viscoelasticity and unique yield-stress properties that allow immediate harvest of embedded cells through a flick of the wrist. This characteristic makes Trpzip hydrogels amenable to syringe extrusion, which we demonstrate with examples of cell delivery and bioprinting. Trpzip hydrogels display innate bioactivity, allowing propagation of human intestinal organoids with apical-basal polarization. Considering these extensive attributes, we anticipate the Trpzip motif will prove a versatile building block for supramolecular assembly of soft materials for biotechnology and medicine.

Magnetic Nanofibrous Hydrogels for Dynamic Control of Stem Cell Differentiation.

The extracellular matrix in tissue consists of complex heterogeneous soft materials with hierarchical structure and dynamic mechanical properties dictating cell and tissue level function. In many natural matrices, there are nanofibrous structures that serve to guide cell activity and dictate the form and function of tissue. Synthetic hydrogels with integrated nanofibers can mimic the structural properties of native tissue; however, model systems with dynamic mechanical properties remain elusive. Here we demonstrate modular nanofibrous hydrogels that can be reversibly stiffened in response to applied magnetic fields. Iron oxide nanoparticles were incorporated into gelatin nanofibers through electrospinning, followed by chemical stabilization and fragmentation. These magnetoactive nanofibers can be mixed with virtually any hydrogel material and reversibly stiffen the matrix at a low fiber content (≤3%). In contrast to previous work, where a large quantity of magnetic material disallowed cell encapsulation, the low nanofiber content allows matrix stiffening with cells in 3D. Using adipose derived stem cells, we show how nanofibrous matrices are beneficial for both osteogenesis and adipogenesis, where stiffening the hydrogel with applied magnetic fields enhances osteogenesis while discouraging adipogenesis. Skeletal myoblast progenitors were used as a model of tissue morphogenesis with matrix stiffening augmenting myogenesis and multinucleated myotube formation. The ability to reversibly stiffen fibrous hydrogels through magnetic stimulation provides a useful tool for studying nanotopography and dynamic mechanics in cell culture, with a scope for stimuli responsive materials for tissue engineering.

Gas-modulating microcapsules for spatiotemporal control of hypoxia.

Oxygen is a vital molecule involved in regulating development, homeostasis, and disease. The oxygen levels in tissue vary from 1 to 14% with deviations from homeostasis impacting regulation of various physiological processes. In this work, we developed an approach to encapsulate enzymes at high loading capacity, which precisely controls the oxygen content in cell culture. Here, a single microcapsule is able to locally perturb the oxygen balance, and varying the concentration and distribution of matrix-embedded microcapsules provides spatiotemporal control. We demonstrate attenuation of hypoxia signaling in populations of stem cells, cancer cells, endothelial cells, cancer spheroids, and intestinal organoids. Varying capsule placement, media formulation, and timing of replenishment yields tunable oxygen gradients, with concurrent spatial growth and morphogenesis in a single well. Capsule containing hydrogel films applied to chick chorioallantoic membranes encourages neovascularization, providing scope for topical treatments or hydrogel wound dressings. This platform can be used in a variety of formats, including deposition in hydrogels, as granular solids for 3D bioprinting, and as injectable biomaterials. Overall, this platform's simplicity and flexibility will prove useful for fundamental studies of oxygen-mediated processes in virtually any in vitro or in vivo format, with scope for inclusion in biomedical materials for treating injury or disease.

Defined Microenvironments Trigger In Vitro Gastrulation in Human Pluripotent Stem Cells.

Gastrulation is a stage in embryo development where three germ layers arise to dictate the human body plan. In vitro models of gastrulation have been demonstrated by treating pluripotent stem cells with soluble morphogens to trigger differentiation. However, in vivo gastrulation is a multistage process coordinated through feedback between soluble gradients and biophysical forces, with the multipotent epiblast transforming to the primitive streak followed by germ layer segregation. Here, the authors show how constraining pluripotent stem cells to hydrogel islands triggers morphogenesis that mirrors the stages preceding in vivo gastrulation, without the need for exogenous supplements. Within hours of initial seeding, cells display a contractile phenotype at the boundary, which leads to enhanced proliferation, yes-associated protein (YAP) translocation, epithelial to mesenchymal transition, and emergence of SRY-box transcription factor 17 (SOX17)+ T/BRACHYURY+ cells. Molecular profiling and pathway analysis reveals a role for mechanotransduction-coupled wingless-type (WNT) signaling in orchestrating differentiation, which bears similarities to processes observed in whole organism models of development. After two days, the colonies form multilayered aggregates, which can be removed for further growth and differentiation. This approach demonstrates how materials alone can initiate gastrulation, thereby providing in vitro models of development and a tool to support organoid bioengineering efforts.

In situ formation of osteochondral interfaces through "bone-ink" printing in tailored microgel suspensions.

Osteochondral tissue has a complex hierarchical structure spanning subchondral bone to articular cartilage. Biomaterials approaches to mimic and repair these interfaces have had limited success, largely due to challenges in fabricating composite hard-soft interfaces with living cells. Biofabrication approaches have emerged as attractive methods to form osteochondral analogues through additive assembly of hard and soft components. We have developed a unique printing platform that is able to integrate soft and hard materials concurrently through freeform printing of mineralized constructs within tunable microgel suspensions containing living cells. A library of microgels based on gelatin were prepared, where the stiffness of the microgels and a liquid "filler" phase can be tuned for bioprinting while simultaneously directing differentiation. Tuning microgel stiffness and filler content differentially directs chondrogenesis and osteogenesis within the same construct, demonstrating how this technique can be used to fabricate osteochondral interfaces in a single step. Printing of a rapidly setting calcium phosphate cement, so called "bone-ink" within a cell laden suspension bath further guides differentiation, where the cells adjacent to the nucleated hydroxyapatite phase undergo osteogenesis with cells in the surrounding medium undergoing chondrogenesis. In this way, bone analogues with hierarchical structure can be formed within cell-laden gradient soft matrices to yield multiphasic osteochondral constructs. This technique provides a versatile one-pot biofabrication approach without harsh post-processing which will aid efforts in bone disease modelling and tissue engineering. STATEMENT OF SIGNIFICANCE: This paper demonstrates the first example of a biofabrication approach to rapidly form osteochondral constructs in a single step under physiological conditions. Key to this advance is a tunable suspension of extracellular matrix microgels that are packed together with stem cells, providing a unique and modular scaffolding for guiding the simultaneous formation of bone and cartilage tissue. The physical properties of the suspension allow direct writing of a ceramic "bone-ink", resulting in an ordered structure of microscale hydrogels, living cells, and bone mimics in a single step. This platform reveals a simple approach to making complex skeletal tissue for disease modelling, with the possibility of repairing and replacing bone-cartilage interfaces in the clinic using a patient's own cells.

Cell-Laden Gradient Microgel Suspensions for Spatial Control of Differentiation During Biofabrication.

During tissue development, stem and progenitor cells form functional tissue with high cellular diversity and intricate micro- and macro-architecture. Current approaches have attempted to replicate this process with materials cues or through spontaneous cell self-organization. However, cell-directed and materials-directed organization are required simultaneously to achieve biomimetic structure and function. Here, it is shown how integrating live adipose derived stem cells with gradient microgel suspensions steers divergent differentiation outcomes. Microgel matrices composed of small particles are found to promote adipogenic differentiation, while larger particles fostered increased cell spreading and osteogenic differentiation. Tuning the matrix formulation demonstrates that early cell adhesion and spreading dictate differentiation outcome. Combining small and large microgels into gradients spatially directs proliferation and differentiation over time. After 21 days of culture, osteogenic conditions foster significant mineralization within the individual microgels, thereby providing cell-directed changes in composition and mechanics within the gradient porous scaffold. Freeform printing of high-density cell suspensions is performed across these gradients to demonstrate the potential for hierarchical tissue biofabrication. Interstitial porosity influences cell expansion from the print and microgel size guides spatial differentiation, thereby providing scope to fabricate tissue gradients at multiple scales through integrated and printed cell populations.

Heterotypic tumor models through freeform printing into photostabilized granular microgels.

The tissue microenvironment contains a complex assortment of multiple cell types, matrices, and vessel structures, which is difficult to reconstruct in vitro. Here, we demonstrate model tumor microenvironments formed through direct writing of vasculature channels and tumor cell aggregates, within a cell-laden microgel matrix. Photocrosslinkable microgels provide control over local and global mechanics, while enabling the integration of virtually any cell type. Direct writing of a Pluronic sacrificial ink into a stromal cell-microgel suspension is used to form vessel structures for endothelialization, followed by printing of melanoma aggregates. Tumor cells migrate into the prototype vessels as a function of spatial location, thereby providing a measure of invasive potential. The integration of perfusable channels with multiple spatially defined cell types provides new avenues for modelling development and disease, with scope for both fundamental research and drug development efforts.

Geometric regulation of histone state directs melanoma reprogramming.

Malignant melanoma displays a high degree of cellular plasticity during disease progression. Signals in the tumor microenvironment are believed to influence melanoma plasticity through changes in the epigenetic state to guide dynamic differentiation and de-differentiation. Here we uncover a relationship between geometric features at perimeter regions of melanoma aggregates, and reprogramming to a stem cell-like state through histone marks H3K4Me2 and H3K9Ac. Using an in vitro tumor microengineering approach, we find spatial enrichment of these histone modifications with concurrent expression of stemness markers. The epigenetic modifier PRDM14 overlaps with H3K9Ac and shows elevated expression in cells along regions of perimeter curvature. siRNA knockdown of PRDM14 abolishes the MIC phenotype suggesting a role in regulating melanoma heterogeneity. Our results suggest mechanotransduction at the periphery of melanoma aggregates may orchestrate the activity of epigenetic modifiers to regulate histone state, cellular plasticity, and tumorigenicity.

Geometrically Structured Microtumors in 3D Hydrogel Matrices.

During cancer progression, a growing tumor encounters variation in the surrounding microenvironment leading to a diverse landscape at the tumor-matrix interface. Topological cues at the interface are believed to influence invasive characteristics; however, most laboratory models involve tumor spheroids that develop a uniform geometry within a homogenous hydrogel. In this communication, a method for templating hydrogels in well-defined 3D architectures is reported. Using melanoma as a model cancer, fabrication of geometrically structured model tumors in a myriad of shapes and sizes is demonstrated. These microtumors can be encapsulated in virtually any polymeric matrix, with demonstrations using poly(ethylene glycol) and gelatin-based hydrogels. Light sheet imaging reveals uniform viability throughout with regions of high curvature at the periphery influencing cellular heterogeneity. These hydrogel encapsulated microtumors can be harvested and implanted in animal models, providing a unique xenograft system where relationships between geometry, progression, and invasion may be systematically studied.

Enzyme Responsive Inverse Opal Hydrogels.

Structured color in nature is controlled by nano- and micro-structured interfaces giving rise to a photonic bandgap. This study presents a biomimetic optical material based on polymeric inverse opals that respond to enzyme activity. Polymer colloids provide a template in which acryloyl-functionalized poly(ethylene glycol) is integrated; dissolution of the colloids leads to a hydrogel inverse opal that can be lithographically patterned using transfer printing. Incorporating enzyme substrates within the voids provides a material that responds to the presence of proteases through a shift in the optical properties.

The structural fate of lipid nanoparticles in the extracellular matrix.

Drug-loaded liposomes are the most successful nanomedicine to date, with multiple FDA-approved systems for a myriad of diseases. While liposome circulation time in blood and retention in tissues have been studied in detail, the structural fate of liposomes-and nanoparticles in general-in the body has not been extensively investigated. Here, we explore the interactions of liposomes with synthetic and natural hydrogel materials to understand how the natural extracellular matrix influences liposome structural characteristics. Small angle X-ray scattering, confocal microscopy, and cryogenic transmission electron microscopy data demonstrate that poly(ethylene glycol) (PEG), gelatin, alginate, and Matrigel® hydrogels cause 200-nm liposomes of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) to transform into micrometer-sized aggregates. These aggregates are composed of multilamellar vesicles around 100 nm in diameter with a mean interlamellar separation of 5.5 nm. Protecting the liposomes with a corona of PEG damps this restructuring effect, making the multilamellar vesicles less stable. We attribute this unilamellar to multilamellar transition to an osmotic driving force from the hydrogel environment. This lipid restructuring has broad ramifications in the design and use of nanomedicines, and in understanding the fate and function of natural lipid-based materials within the tissue microenvironment.