Centering Single Cells in Microgels via Delayed Crosslinking Supports Long-Term 3D Culture by Preventing Cell Escape

Abstract

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.

Single-cell RNA-seq of rheumatoid arthritis synovial tissue using low cost microfluidic instrumentation

“…In contrast, the platform presented here is easy to use and can be implemented for a variety of droplet microfluidic (partitioning) or continuous phase microfluidic based experiments. Potential applications of this system include recent work profiling immune repertoires from hundreds of thousands of single cells 13 and combined single-cell transcriptome and epitope profiling 14 in addition to ddPCR 15 , ddMDA 16 , hydrogel microsphere fabrication for 3D cell culture 17,18 , chemical microfluidic gradient generation 19 and microparticle size sorting [20][21][22] . The instrument is comprised of electronic and pneumatic components affixed to a 3D printed frame.…”

Biopolymer Microparticles Prepared by Microfluidics for Biomedical Applications

“…Meanwhile, another type of dextran microspheres was developed for single cell encapsulation through a diffusion-based enzymatic crosslinking of tyramine-conjugated dextran in microfluidically generated droplets. [174] Single MSCs encapsulated in the dextran microspheres exhibited almost perfect centering without a significant escape from the center during 7 days of culture. In addition, they demonstrated a high long-term viability and multilineage differentiation capacity even after 28 days of culture.…”

“…Two hydrophobic anticancer drugs, celecoxib and sorafenib, were encapsulated in the dextran hollow MPs with a high loading efficiency and were released simultaneously by virtue of the dissolution of dextran matrix in a controlled manner, indicating the applicability for multidrug delivery. Meanwhile, another type of dextran microspheres was developed for single cell encapsulation through a diffusion‐based enzymatic crosslinking of tyramine‐conjugated dextran in microfluidically generated droplets . Single MSCs encapsulated in the dextran microspheres exhibited almost perfect centering without a significant escape from the center during 7 days of culture.…”

Spatially and temporally controlled hydrogels for tissue engineering

“…Recent innovations even allowed for microfluidic encapsulation of individual cells in microgels that are only marginally larger than the cell they contain [66]. Spatial placement of the cell in these microgels is of high importance to prevent cell egression; it has been reported that delayed on-chip gelation places cells in the microgel’s center, which enables long-term culture [67]. Frequently, hydrophilic hydrogel precursor solutions are co-flown with a hydrophobic liquid, such as oils in a microfluidic channel to generate microdroplets of liquid hydrogel precursor.…”