Mass production of lumenogenic human embryoid bodies and functional cardiospheres using in-air-generated microcapsules

Abstract

Organoids are engineered 3D miniature tissues that are defined by their organ-like structures, which drive a fundamental understanding of human development. However, current organoid generation methods are associated with low production throughputs and poor control over size and function including due to organoid merging, which limits their clinical and industrial translation. Here, we present a microfluidic platform for the mass production of lumenogenic embryoid bodies and functional cardiospheres. Specifically, we apply triple-jet in-air microfluidics for the ultra-high-throughput generation of hollow, thin-shelled, hydrogel microcapsules that can act as spheroid-forming bioreactors in a cytocompatible, oil-free, surfactant-free, and size-controlled manner. Uniquely, we show that microcapsules generated by in-air microfluidics provide a lumenogenic microenvironment with near 100% efficient cavitation of spheroids. We demonstrate that upon chemical stimulation, human pluripotent stem cell-derived spheroids undergo cardiomyogenic differentiation, effectively resulting in the mass production of homogeneous and functional cardiospheres that are responsive to external electrical stimulation. These findings drive clinical and industrial adaption of stem cell technology in tissue engineering and drug testing.

Photoannealing of Microtissues Creates High‐Density Capillary Network Containing Living Matter in a Volumetric‐Independent Manner

“…By tuning the cell concentration in the ATA polymer solution, the number of cells within the engineered microtissues can be accurately controlled, resulting in 7 ± 2, 15 ± 4, 30 ± 5, 39 ± 9, and 56 ± 9 cells per microgel when using concentrations of 1 × 10 6 , 3 × 10 6 , 5 × 10 6 , 7 × 10 6 , and 10 7 cells mL −1 , respectively (Figure 3b,c). Importantly, although ethanol is initially used to facilitate the homogenous in‐air cross‐linking of the microgels, ethanol does not pose severe cytotoxicity to cells due to the short in‐flight contact time with ethanol (≈100 ms) before it is diluted in an excess of culture medium to a concentration of ≥1% which has been reported as nontoxic for exposure times below 24 h. [ 41 ] As a result, both the ionic cross‐link used for microgel formation, and the visible‐light‐initiated radical reaction used for PALM are associated with a low cytotoxicity as evidenced by high cell viability (e.g., >80%) (Figure 3d,e). Importantly, using culture medium as the solvent did not negatively affect the photoannealing process (Figure S5, Supporting Information).…”

“…Alginate was selected as the base material due to its compatibility with in-air microfluidics, which necessitates a rapid (subsecond) cross-linking. [39][40][41] This droplet train collided with a second liquid jet containing calcium chloride, which effectively wraps around each droplet via surface tension-driven Marangoni flow, and subsequently ionically cross-linked the alginate in the droplet to form a microgel. To ensure homogenous cross-linking of the alginate droplets, the surface tension of the second jet was lowered by the addition of a small amount of ethanol (10% v/v) to match the rapid ionic crosslinking during the ≈100 ms flight time before droplet collection.…”

Large-scale generation of uniform sub-100 μm adipocyte spheroids in hydrogel microcapsules using a flow-focusing microfluidic device

“…While microwell platforms have expanded the number of spheroids per plate to a few thousand, the number produced is still insufficient for high-throughput screening (HTS), and achieving size uniformity is challenging due to inconsistencies in cell densities per microwell (Kim et al, 2021; Oka et al, 2019). Alternatively, microfluidic technologies facilitate the high-throughput production of spheroids within emulsions or microcapsules (Chan et al, 2013; Liu et al, 2020; van Loo et al, 2023); however, no one has yet successfully cultured adipocyte spheroids using these methods.…”