Single cell-laden protease-sensitive microniches for long-term culture in 3D

Abstract

Single cell-laden three-dimensional (3D) microgels that can serve to mimic stem cell niches in vitro, and are therefore termed microniches, can be efficiently fabricated by droplet-based microfluidics. In this technique an aqueous polymer solution along with a highly diluted cell solution is injected into a microfluidic device to create monodisperse pre-microgel droplets that are then solidified by a polymer crosslinking reaction to obtain monodisperse single cell-laden microniches. However, problems limiting this approach studying the fate of single cells include Poisson encapsulation statistics that result in mostly empty microniches, and cells egressing from the microniches during subsequent cell culture. Here, we present a strategy to bypass Poisson encapsulation statistics in synthetic microniches by selective crosslinking of only cell-laden pre-microgel droplets. Furthermore, we show that we can position cells in the center of the microniches, and that even in protease-sensitive microniches this greatly reduces cell egress. Collectively, we present the development of a versatile protocol that allows for unprecedented efficiency in creation of synthetic protease-sensitive microniches for probing single stem cell fate in 3D.

Long‐Term Perfusion Culture of Monoclonal Embryonic Stem Cells in 3D Hydrogel Beads for Continuous Optical Analysis of Differentiation

“…For the majority of trapped cells (71%, 17 of 24) growth, proliferation, and spheroid formation could be observed (Figure ; Video S3, Supporting Information), matching the clonogenic efficiency observed in adherent cultures . Similar to poly(ethylene glycol) (PEG)‐based gels, 13% of cells (3 of 24) migrated through the hydrogel and escaped from the agarose bead (Video S4, Supporting Information) and 17% (4 of 24) did not survive the first 15 h and were not able to form a spheroid (Video S5, Supporting Information).…”

Single-Cell Microgels: Technology, Challenges, and Applications

“…The importance of bioactive components in promoting cellular function was underlined in a recent study by Leijten and coworkers, where the lineage commitment of individually encapsulated mesenchymal stem cells was steered from an adipogenic to an osteogenic fate by enriching bioinert dextran microgels with bioactive hyaluronic acid polymer groups [21]. Microgel stiffness has also been demonstrated to exert a profound effect on the differentiation of individual encapsulated stem cells in cell-adhesive and enzymatically degradable microgels [22], which corroborates previous work in bulk hydrogels [23]. Additional functionality comes from tuning the microgel mesh size.…”

Emerging Trends in Information‐Driven Engineering of Complex Biological Systems

“…[95] Generally, photopolymerization chemistries rely on a photoinitiator that forms radicals upon illumination, which polymerizes a monomer that possesses multifunctional crosslinkers. [102] Additionally, PEG has been demonstrated to be incredibly versatile and amenable to modification with various GAGs to produce GAG composites with tunable properties, which offers a potential route to the critical role of these polysaccharides in ECM biology (reviewed elsewhere). [96] Photopolymerization techniques are widely applied to hydrogels and other polymers for tissue engineering applications and have been reviewed in details elsewhere.…”