Modular bioinks based on single cell microgels within distinct injectable prepolymers enable uncoupling of biomaterials' micro‐ and macroenvironments. These inks allow biofabrication of 3D constructs ...that recapitulate the multiscale modular design of native tissues with a single cell resolution. This approach represents a major step forward in endowing engineered constructs with the multifunctionality that underlies the behavior of native tissues.
Spatiotemporal control over engineered tissues is highly desirable for various biomedical applications as it emulates the dynamic behavior of natural tissues. Current spatiotemporal biomaterial ...functionalization approaches are based on cytotoxic, technically challenging, or non-scalable chemistries, which has hampered their widespread usage. Here we report a strategy to spatiotemporally functionalize (bio)materials based on competitive supramolecular complexation of avidin and biotin analogs. Specifically, an injectable hydrogel is orthogonally post-functionalized with desthiobiotinylated moieties using multivalent neutravidin. In situ exchange of desthiobiotin by biotin enables spatiotemporal material functionalization as demonstrated by the formation of long-range, conformal, and contra-directional biochemical gradients within complex-shaped 3D hydrogels. Temporal control over engineered tissue biochemistry is further demonstrated by timed presentation and sequestration of growth factors using desthiobiotinylated antibodies. The method's universality is confirmed by modifying hydrogels with biotinylated fluorophores, peptides, nanoparticles, enzymes, and antibodies. Overall, this work provides a facile, cytocompatible, and universal strategy to spatiotemporally functionalize materials.
Abstract
Organoids are engineered 3D miniature tissues that are defined by their organ-like structures, which drive a fundamental understanding of human development. However, current organoid ...generation methods are associated with low production throughputs and poor control over size and function including due to organoid merging, which limits their clinical and industrial translation. Here, we present a microfluidic platform for the mass production of lumenogenic embryoid bodies and functional cardiospheres. Specifically, we apply triple-jet in-air microfluidics for the ultra-high-throughput generation of hollow, thin-shelled, hydrogel microcapsules that can act as spheroid-forming bioreactors in a cytocompatible, oil-free, surfactant-free, and size-controlled manner. Uniquely, we show that microcapsules generated by in-air microfluidics provide a lumenogenic microenvironment with near 100% efficient cavitation of spheroids. We demonstrate that upon chemical stimulation, human pluripotent stem cell-derived spheroids undergo cardiomyogenic differentiation, effectively resulting in the mass production of homogeneous and functional cardiospheres that are responsive to external electrical stimulation. These findings drive clinical and industrial adaption of stem cell technology in tissue engineering and drug testing.
Living microtissues are used in a multitude of applications as they more closely resemble native tissue physiology, as compared to 2D cultures. Microtissues are typically composed of a combination of ...cells and materials in varying combinations, which are dictated by the applications’ design requirements. Their applications range wide, from fundamental biological research such as differentiation studies to industrial applications such as cruelty-free meat production. However, their translation to industrial and clinical settings has been hindered due to the lack of scalability of microtissue production techniques. Continuous microfluidic processes provide an opportunity to overcome this limitation as they offer higher throughput production rates as compared to traditional batch techniques, while maintaining reproducible control over microtissue composition and size. In this review, we provide a comprehensive overview of the current approaches to engineer microtissues with a focus on the advantages of, and need for, the use of continuous processes to produce microtissues in large quantities. Finally, an outlook is provided that outlines the required developments to enable large-scale microtissue fabrication using continuous processes.
•This review provides an overview of techniques used to create microtissues, and their applications in research and industry.•Batch production methods are compared to continuous production methods in terms of throughput and translatability.•Continuous production methods are ideal for many applications related to clinical translation and industrial valorization.•Optimization of continuous production methods is required to advance translation of microtissues to clinical/industrial use.
Human in vitro models of neural tissue with tunable microenvironment and defined spatial arrangement are needed to facilitate studies of brain development and disease. Towards this end, embedded ...printing inside granular gels holds great promise as it allows precise patterning of extremely soft tissue constructs. However, granular printing support formulations are restricted to only a handful of materials. Therefore, there has been a need for novel materials that take advantage of versatile biomimicry of bulk hydrogels while providing high‐fidelity support for embedded printing akin to granular gels. To address this need, Authors present a modular platform for bioengineering of neuronal networks via direct embedded 3D printing of human stem cells inside Self‐Healing Annealable Particle‐Extracellular matrix (SHAPE) composites. SHAPE composites consist of soft microgels immersed in viscous extracellular‐matrix solution to enable precise and programmable patterning of human stem cells and consequent generation mature subtype‐specific neurons that extend projections into the volume of the annealed support. The developed approach further allows multi‐ink deposition, live spatial and temporal monitoring of oxygen levels, as well as creation of vascular‐like channels. Due to its modularity and versatility, SHAPE biomanufacturing toolbox has potential to be used in applications beyond functional modeling of mechanically sensitive neural constructs.
In this study, a modular platform for bioengineering of neuronal networks via direct embedded 3D printing of human stem cells inside self‐healing annealable particle‐extracellular matrix (SHAPE) composites is presented. The approach allows freeform patterning and functional maturation of human neural constructs with potential for both tissue engineering and disease modeling.
Engineered living microtissues such as cellular spheroids and organoids have enormous potential for the study and regeneration of tissues and organs. Microtissues are typically engineered via ...self‐assembly of adherent cells into cellular spheroids, which are characterized by little to no cell–material interactions. Consequently, 3D microtissue models currently lack structural biomechanical and biochemical control over their internal microenvironment resulting in suboptimal functional performance such as limited stem cell differentiation potential. Here, this work report on stimuli‐responsive cell‐adhesive micromaterials (SCMs) that can self‐assemble with cells into 3D living composite microtissues through integrin binding, even under serum‐free conditions. It is demonstrated that SCMs homogeneously distribute within engineered microtissues and act as biomechanically and biochemically tunable designer materials that can alter the composite tissue microenvironment on demand. Specifically, cell behavior is controlled based on the size, stiffness, number ratio, and biofunctionalization of SCMs in a temporal manner via orthogonal secondary crosslinking strategies. Photo‐based mechanical tuning of SCMs reveals early onset stiffness‐controlled lineage commitment of differentiating stem cell spheroids. In contrast to conventional encapsulation of stem cell spheroids within bulk hydrogel, incorporating cell‐sized SCMs within stem cell spheroids uniquely provides biomechanical cues throughout the composite microtissues’ volume, which is demonstrated to be essential for osteogenic differentiation.
Cellular spheroids are used to mimic native tissues but only offer poor control over their internal microenvironment. Here, stimuli‐responsive cell‐adhesive micromaterials (SCMs) that interact and self‐assemble with cells into 3D living composite tissues are developed. The behavior of stem cell spheroids, including their lineage commitment, is controlled from within by tuning cell–material interactions via the amount, size, and temporal stiffness of SCMs.
Modular bioinks based on single‐cell‐laden microgels are engineered using droplet microfluidics and leveraged to uncouple micro‐ and macroenvironments of biomaterials by Marcel Karperien and ...co‐workers in article 1600913. These inks enable straightforward biofabrication of 3D constructs that recapitulate the multiscale modular design of native tissues with a single cell resolution. This approach represents a major step forward in endowing engineered constructs with the multifunctionality that underlies the behavior of native tissues.
Single-cell-laden microgels effectively act as the engineered counterpart of the smallest living building block of life: a cell within its pericellular matrix. Recent breakthroughs have enabled the ...encapsulation of single cells in sub-100-μm microgels to provide physiologically relevant microniches with minimal mass transport limitations and favorable pharmacokinetic properties. Single-cell-laden microgels offer additional unprecedented advantages, including facile manipulation, culture, and analysis of individual cell within 3D microenvironments. Therefore, single-cell microgel technology is expected to be instrumental in many life science applications, including pharmacological screenings, regenerative medicine, and fundamental biological research. In this review, we discuss the latest trends, technical challenges, and breakthroughs, and present our vision of the future of single-cell microgel technology and its applications.
Several recent technological breakthroughs have facilitated and improved the manufacturing of single-cell-laden microgels. In particular, microfluidic technologies have enabled the high-yield and long-term encapsulation of individual cells in microgels.
Encapsulating individual cells into micrometer-sized hydrogels (i.e., microgels) provides user-defined 3D culture conditions that can be leveraged to advance single-cell analysis platforms, cell-based tissue engineering, and regenerative medicine applications.
Early adopters are currently developing and implementing various high-throughput micromanufacturing techniques that will empower the clinical and industrial translation of single-cell-laden microgels as versatile high-resolution 3D cellular microniches for numerous biotechnological applications.
PDF
