Abstract
Advances in the fields of biofabrication, microfluidics, and sensor technologies have enabled bioreactors to create and monitor in vitro environments with increasing similarity to the native niche in vivo. This thesis addressed the current disparity between study throughput and tissue/bioreactor complexity through design and development of an extensible bioreactor for mechanically-loaded, individually-addressable, dually-perfused, biofabricated tissue constructs for both mechanistic studies (via sample recovery) and high-throughput screening (via in situ monitoring and assessment). Furthermore, this bioreactor was applied to musculoskeletal tissue engineering research through the creation of well inserts supporting cast cell-laden hydrogel constructs for null- and selective-crosstalk between the two media channels.
The bioreactor housed samples in a 3D-printed, ANSI/SLAS-compliant 96-well microplate with clear-bottomed wells to maintain compatibility with existing imaging modalities and laboratory robotics. Each well (6x10 mm) was provided two independent perfusion flow circuits and uniaxial compressive loading. Additive manufacturing, precision machining, and diffusion bonding were employed to create flow channels to each well of the 96-well microplate. The microplate interfaced with bioreactor modules responsible for fluid transport and mechanical loading through a docking station. Fluid was driven by an autoclavable peristaltic pump. Media was stored in standard deep-well microplates capped with SLA-printed lids. Mechanical loading was applied through a flexible diaphragm by a computer-controlled pneumatic valve to modulate an air source. The bioreactor materials in the wetted path were validated as non-cytotoxic according to ISO 10993: Part 5. The mechanical design was validated through extended user interaction testing with proof-of-concept experiments on a 3D ovarian cancer coculture model in gelatin-methacryloyl (gel-MA) hydrogels to identify and eliminate obstacles to reliable scalability. Additionally, the in situ monitoring features were validated for fluorescent microscopy, x-ray imaging, perfused particle flow tracing, and quantification of metabolic activity in a perfused system. The bioreactor was extended to a platform to support musculoskeletal research, starting at a vascular microphysiological system (MPS), i.e. a bioreactor populated with perfused vascular tissue constructs. These constructs relied on artificial channels to assist cellular self-assembly to form vascular networks in gelatin-norbornene (gel-NOR) hydrogels crosslinked with visible-light photoinitiators. The bioreactor supported in situ biofabrication via bioassembly or casting-based strategies. However, the sample well imposed strict constraints on the bioprinter and materials. An SLA-printed well insert was developed to provide an alternative method for construct production with minimal user interaction. The insert, too, relied on artificial channels and cellular self-assembly in cast gel-NOR hydrogels. This work culminated in the validation of the MPS experimental protocol using a multi-flow study as a model. The microplate-based sample housing and reservoirs simplified user interaction and minimized risk contamination, while the vascular well inserts were demonstrated to be well-suited for cryosectioning and fluorescent imaging, necessary for future immunohistofluorescence evaluations of vascular network maturity.
As a result of this work, the scientific community has access to a validated platform to support high-throughput studies toward development of vascularized osteochondral tissue models, and a bioreactor inherently flexible to apply to a range of applications, up to and including pharmacokinetic/pharmacodynamic studies for drug development.