A 96-well microplate bioreactor platform supporting individual dual perfusion and high-throughput assessment of simple or biofabricated 3D tissue models

Abstract

Traditional 2D monolayer cell cultures and submillimeter 3D tissue construct cultures used widely in tissue engineering are limited in their ability to extrapolate experimental data to predict in vivo responses due to their simplistic organization and lack of stimuli. The rise of biofabrication and bioreactor technologies has sought to address this through the development of techniques to spatially organize components of a tissue construct, and devices to supply these tissue constructs with an increasingly in vivo-like environment. Current bioreactors supporting both parenchymal and barrier tissue constructs in interconnected systems for body-on-a-chip platforms have chosen to emphasize study throughput or system/tissue complexity. Here, we report a platform to address this disparity in throughput and both system complexity (by supporting multiple in situ assessment methods) and tissue complexity (by adopting a construct-agnostic format). We introduce an ANSI/SLAS-compliant microplate and docking station fabricated via stereolithography (SLA), or precision machining, to provide up to 96 samples (Ø6 × 10 mm) with two individually-addressable fluid circuits (192 total), loading access, and inspection window for imaging during perfusion. Biofabricated ovarian cancer models were developed to demonstrate the in situ assessment capabilities via microscopy and a perfused resazurin-based metabolic activity assay. In situ microscopy highlighted flexibility of the sample housing to accommodate a range of sample geometries. Utility for drug screening was demonstrated by exposing the ovarian cancer models to an anticancer drug (doxorubicin) and generating the dose-response curve in situ, while achieving an assay quality similar to static wellplate culture. The potential for quantitative analysis of temporal tissue development and screening studies was confirmed by imaging soft- (gelatin) and hard-tissue (calcium chloride) analogs inside the bioreactor via spectral computed tomography (CT) scanning. As a proof-of-concept for particle tracing studies, flowing microparticles were visualized to inform the design of hydrogel constructs. Finally, the ability for mechanistic yet high-throughput screening was demonstrated in a vascular coculture model adopting endothelial and mesenchymal stem cells (HUVEC-MSC), encapsulated in gelatin-norbornene (gel-NOR) hydrogel cast into SLA-printed well inserts. This study illustrates the potential of a scalable dual perfusion bioreactor platform for parenchymal and barrier tissue constructs to support a broad range of multi-organ-on-a-chip applications.

Visible Light Cross‐Linking of Gelatin Hydrogels Offers an Enhanced Cell Microenvironment with Improved Light Penetration Depth

“…Although we did observe that the UV + I2959 system had a faster cross‐linking rate compared to both the Vis + LAP and Vis + Ru/SPS systems, the mass loss and swelling studies were conducted in an ideal environment without taking oxygen inhibition and light penetration depth into account, where the macromer was irradiated while sandwiched between a glass slide and cover slip, and is not an accurate representation of the downstream application. Moreover, in addition to Gel‐MA hydrogels alone, we have successfully employed this visible light system to other polymers including heparin, hyaluronic acid, poly(vinyl alcohol), and gellan gum, all of which were functionalized with unsaturated vinyl moieties, such as methacryloyl or allyl groups . Taking these factors into account, our work suggests that both synthetic and biological polymers modified with functional vinyl moieties can be cross‐linked through different chemistries such as chain‐growth methacryloyl or step‐growth thiol–ene photo‐click polymerization, using Vis + Ru/SPS.…”

“…Moreover, in addition to Gel-MA hydrogels alone, we have successfully employed this visible light system to other polymers including heparin, hyaluronic acid, poly(vinyl alcohol), and gellan gum, all of which were functionalized with unsaturated vinyl moieties, such as methacryloyl or allyl groups. [36,38,51] Taking these factors into account, our work suggests that both synthetic and biological polymers modified with functional vinyl moieties can be cross-linked through different chemistries such as chain-growth methacryloyl or step-growth thiol-ene photo-click polymerization, using Vis + Ru/SPS.…”

Redox‐Active Dynamic Self‐Supporting Thixotropic 3D‐Printable G‐Quadruplex Hydrogels

“…[1][2][3][4][5] Hydrogels with injectable and self-healing properties restoret heir inherent elastic nature and possess quick gel-solgel transitions. Supramolecular hydrogels are appliedi nv arious fields including woundh ealing, tissue engineering, nanotechnology and sensing applications.…”

“…Supramolecular hydrogels are appliedi nv arious fields including woundh ealing, tissue engineering, nanotechnology and sensing applications. [1][2][3][4][5] Hydrogels with injectable and self-healing properties restoret heir inherent elastic nature and possess quick gel-solgel transitions. Fast gel-sol-gel transitions lead to aw ide range of applications including 3D printing, drug release,e lectromagnetic shielding and supercapacitors.…”

A Perfused Microfluidic System to Study the Differentiation of Neural Stem Cells in vitro

“…A perfused culture can constantly provide continuous nutrient supply as well as removing the waste, thus maintaining a fresh and stable local microenvironment. Some perfused culture systems have been developed for use for either cell expansion [Okamoto et al, 2013;Chapman, 2014] or three-dimensional cell culture [Parrish et al, 2018;Vormann et al, 2018].…”