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.

Biofabrication commonly involves the use of liquid droplets to transport cells to the printed structure. However, the viability of the cells after impact is poorly controlled and understood, hampering applications including cell spraying, inkjet bioprinting, and laser-assisted cell transfer. Here, we present an analytical model describing the cell viability after impact as a function of the cell-surrounding droplet characteristics. The model connects (1) the cell survival as a function of cell membrane elongation, (2) the membrane elongation as a function of the cell-containing droplet size and velocity, and (3) the substrate properties. The model is validated by cell viability measurements in cell spraying, which is a method for biofabrication and used for the treatment of burn wounds. The results allow for rational optimization of any droplet-based cell deposition technology, and we include practical suggestions to improve the cell viability in cell spraying.

Single-cell-laden microgels support physiological 3D culture conditions while enabling straightforward handling and high-resolution readouts of individual cells. However, their widespread adoption for long-term cultures is limited by cell escape. In this work, it is demonstrated that cell escape is predisposed to off-center encapsulated cells. High-speed microscopy reveals that cells are positioned at the microgel precursor droplets' oil/water interface within milliseconds after droplet formation. In conventional microencapsulation strategies, the droplets are typically gelled immediately after emulsification, which traps cells in this off-center position. By delaying crosslinking, driving cells toward the centers of microgels is succeeded. The centering of cells in enzymatically crosslinked microgels prevents their escape during at least 28 d. It thereby uniquely enables the long-term culture of individual cells within <5-µm-thick 3D uniform hydrogel coatings. Single cell analysis of mesenchymal stem cells in enzymatically crosslinked microgels reveals unprecedented high cell viability (>90%), maintained metabolic activity (>70%), and multilineage differentiation capacity (>60%) over a period of 28 d. The facile nature of this microfluidic cell-centering method enables its straightforward integration into many microencapsulation strategies and significantly enhances control, reproducibility, and reliability of 3D single cell cultures.

Nanoemulsion-induced enzymatic crosslinking of tyramine-functionalized polymer dropletsKamperman Important noteTo cite this publication, please use the final published version (if applicable). Please check the document version above. CopyrightOther than for strictly personal use, it is not permitted to download, forward or distribute the text or part of it, without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license such as Creative Commons. Takedown policyPlease contact us and provide details if you believe this document breaches copyrights. We will remove access to the work immediately and investigate your claim. AbstractIn situ gelation of water-in-oil polymer emulsions is a key method to produce hydrogel particles.Although this approach is in principle ideal for encapsulating bioactive components such as cells, the oil phase can interfere with straightforward presentation of crosslinker molecules. Several approaches have been developed to induce in-emulsion gelation by exploiting the triggered generation or release of crosslinker molecules. However, these methods typically rely on photo-or acid-based reactions that are detrimental to cell survival and functioning. In this work, we demonstrate the diffusion-based supplementation of small molecules for the in-emulsion gelation of multiple tyramine-functionalized polymers via enzymatic crosslinking using a H2O2/oil nanoemulsion. This strategy is compatible with various emulsification techniques, thereby readily supporting the formation of monodisperse hydrogel particles spanning multiple length scales ranging from the nano-to the millimeter. As proof of principle, we leveraged droplet microfluidics in combination with the cytocompatible nature of enzymatic crosslinking to engineer hollow cellladen hydrogel microcapsules that support the formation of viable and functional 3D microtissues.The straightforward, universal, and cytocompatible nature of nanoemulsion-induced enzymatic crosslinking facilitates its rapid and widespread use in numerous food, pharma, and life science applications.

Cell-laden micrometer-sized hydrogels (microgels) hold great promise for improving high throughput ex-vivo drug screening and engineering biomimetic tissues. Microfluidics is a powerful tool to produce microgels. However, only a limited amount of biomaterials have been reported to be compatible with on-chip microgel formation. Moreover, these biomaterials are often associated with mechanical instability, cytotoxicity, and cellular senescence. To resolve this challenge, dextran-tyramine has been explored as a novel biomaterial for on-chip microgel formation. In particular, dextran-tyramine is compared with two commonly used biomaterials, namely, polyethylene-glycol diacrylate (PEGDA) and alginate, which crosslink through enzymatic reaction, UV polymerization, and ionic interaction, respectively. Human mesenchymal stem cells (hMSCs) encapsulated in dextran-tyramine microgels demonstrate significantly higher (95%) survival as compared to alginate (81%) and PEGDA (69%). Long-term cell cultures demonstrate that hMSCs in PEGDA microgels become senescent after 7 d. Alginate microgels dissolve within 7 d due to Ca loss. In contrast, dextran-tyramine based microgels remain stable, sustain hMSCs metabolic activity, and permit for single-cell level analysis for at least 28 d of culture. In conclusion, enzymatically crosslinking dextran-tyramine conjugates represent a novel biomaterial class for the on-chip production of cell-laden microgels, which possesses unique advantages as compared to the commonly used UV and ionic crosslinking biomaterials.