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

Abstract

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.

Spatially and temporally controlled hydrogels for tissue engineering

“…Microfluidic platforms have been used to microfabricate hydrogel spheres with well-defined diameters, architectures, and chemical compositions [60–64], as well as fibers of a particular architecture including hollow fibers and gradients within hydrogels [65]. Numerous studies have demonstrated the generation of cell-laden hydrogel microparticles using microfluidic devices[64]. Recent innovations even allowed for microfluidic encapsulation of individual cells in microgels that are only marginally larger than the cell they contain [66].…”

Nanoemulsion-induced enzymatic crosslinking of tyramine-functionalized polymer droplets

“…remaining PDMS particles from inlet punching) from interfering with the droplet generation or crosslinking processes (Figure 4d). Furthermore, the aqueous phase inlet contained a previously reported pillar structure 13 that ensured particle homogenization to aid evenly distributed encapsulation of particles (e.g. cells) (Figure 4e).…”

“…[8][9][10] In particular, emulsions can be continuously produced at high rates while stabilizing surfactants make them compatible with relatively slow (seconds to minutes) gelation mechanisms such as Michael-type addition, 11 temperature-dependent gelation, 12 and enzyme-based crosslinking approaches. 13 The majority of polymer gelation strategies however requires the presence of crosslinker molecules such as ions and radicals, 14 which is not trivial in emulsions, as these are typically multiphase immiscible systems where oil hampers the direct mixing of the hydrogel precursor and its crosslinker.…”

“…11,15,16 However, this strategy reduces the control over the emulsification process due to increasing liquid viscosity and may result in inhomogeneous polymeric networks because the crosslinking is induced before the hydrogel precursor and its crosslinker are homogeneously mixed. 13 Furthermore, coupling gelation and emulsification frequently causes device clogging and off-center cell encapsulation, which hampers the long-term applications of cell-laden hydrogel particles. 17 Consequently, it is often desirable to sequentially perform the emulsification and gelation processes, which requires the in-emulsion generation or release of crosslinker molecules.…”

“…6,[18][19][20][21] However, the commonly used acid-, photo-, and heat-triggered crosslinking strategies are detrimental to cell survival and function, or are technically challenging as they require the formation of labile crosslinker-laden complexes. 13,22,23 Alternatively, gelation of emulsified hydrogel precursor droplets can be induced via the diffusionbased supplementation of crosslinker molecules, which does not depend on technically challenging or cytotoxic triggers. For example, alginate microspheres have been formed by supplementing the oil phase with crosslinker nanoparticles 24,25 and nanodroplets 26 that diffuse through the oil phase and induce crosslinking of the emulsified polymer droplets.…”