S.J. Henke

Optimizing cell viability in droplet-based cell deposition

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.

Enzymatic Crosslinking of Polymer Conjugates is Superior over Ionic or UV Crosslinking for the On-Chip Production of Cell-Laden Microgels

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 Ca2+ 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.

Nanoemulsion-induced enzymatic crosslinking of tyramine-functionalized polymer droplets

In 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 cell-laden 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.