Micropocket hydrogel devices for all-in-one formation, assembly, and analysis of aggregate-based tissues

Abstract

Multicellular aggregated tissues have grown critically important in benchtop biomedical research, both as stand-alone spheroids and when assembled into larger bioengineered constructs. However, typical systems for aggregate formation are limited in their capacity to reliably handle such cultures at various experimental stages in a broadly accessible, consistent, and scalable manner. In this work, we develop a broadly versatile all-in-one biofabrication strategy to form uniform, spherical, multicellular aggregates that can be maintained at precisely defined positions for analysis or transfer into a larger tissue. The 3D-printed MicroPocket Culture (MPoC) system consists of an array of simple geometrybased valves in a polyacrylamide hydrogel, and is able to produce hundreds of uniformly-sized aggregates in standard tissue culture well plates, using simple tools that are readily available in all standard biological wet-labs. The model breast cancer aggregates formed in these experiments are retained in defined positions on chip during all liquid handling steps required to stimulate, label, and image the experiment, enabling high-throughput studies on this culture model. Furthermore, MPoCs enable robust formation of aggregates in cell types that do not conventionally form such structures. Finally, we demonstrate that this single platform can also be used to generate complex 3D tissues from the precisely-positioned aggregate building blocks. To highlight the unique and broad versatility of this technique, we develop a simple 3D invasion assay and show that cancer cells preferentially migrate towards nearby model tumors; demonstrating the importance of spatial precision when engineering 3D tissues. Together, this platform presents a broadly accessible and uniquely capable system with which to develop advanced aggregate-based models for tissue engineering, fundamental research, and applied drug discovery.

Mapping cellular-scale internal mechanics in 3D tissues with thermally responsive hydrogel probes

“…3a, b), which are commonly used to model three-dimensional, diffusionlimited, and high cell-density tissues 35 . A model T47D cell line suspension was mixed with µTAMs, and formed into spheroids by aggregation 36 . As a first demonstration of µTAM stiffness sensing, we measured sensor expansion before and after tissue crosslinking through paraformaldehyde fixation, which we verified would not affect µTAM operation ( Supplementary Fig.…”

Mapping cellular-scale internal stiffness in 3D tissues with smart material hydrogel probes

“…Based on our calibration and modelling experiments, we elected to use the 3% NiPAAM/0.2% bisacrylamide formulation for all described experiments, as it provides greater measurement sensitivity over the stiffness range expected in spheroids 37,38 . A model T47D cell line was mixed with µTAMs, and formed into spheroids by confinement 39 . As a first demonstration of µTAM stiffness sensing, we measured spheroid rigidities before and after tissue crosslinking through paraformaldehyde fixation, which we verified would not affect µTAM operation ( Supplementary Fig.…”

“…3C) 41 ; and a micropocket-based system in which cells passively settle into and aggregate in hydrogel cavities ( Fig. 3F; Supplementary Fig S5) 39 . These two techniques both rely on cell-driven aggregation and compaction in confined volumes, and should hence produce reasonably similar structures.…”

“…4A, Supplementary Fig. S6) that we have previously established to be non-invasive (T47D) and invasive (MDA-MB-231) in collagen hydrogels over 2 days in culture 39 . The apparent internal stiffness of non-invasive spheroids varied between 0 and 12 kPa.…”

Cost-effective fabrication of photopolymer molds with multi-level microstructures for PDMS microfluidic device manufacture

“…In regard, suspension cells culturing within microuidic devices is challenging because simply renewing the medium can lead to accidental cell loss without a trapping mechanism. 18,19 Yue et al reported the fabrication of multi-level channels for cell quantication and cell culture by using a screen printing method. 20 Moreover, microchannels with trapezoidal cross-section have been reported as a retention mechanism that enabled the capture and suspension culture of mammalian cells through inertial microuidics.…”