Development, characterization, and applications of multi-material stereolithography bioprinting

www.nature.com/scientificreports OPEN Development, characterization, and applications of multi‑material stereolithography bioprinting Bagrat Grigoryan1, Daniel W. Sazer1, Amanda Avila1, Jacob L. Albritton1, Aparna Padhye2, Anderson H. Ta1, Paul T. Greenfield1, Don L. Gibbons2,3 & Jordan S. Miller1* As a 3D bioprinting technique, hydrogel stereolithography has historically been limited in its ability to capture the spatial heterogeneity that permeates mammalian tissues and dictates structure–function relationships. This limitation stems directly from the difficulty of preventing unwanted material mixing when switching between different liquid bioinks. Accordingly, we present the development, characterization, and application of a multi-material stereolithography bioprinter that provides controlled material selection, yields precise regional feature alignment, and minimizes bioink mixing. Fluorescent tracers were first used to highlight the broad design freedoms afforded by this fabrication strategy, complemented by morphometric image analysis to validate architectural fidelity. To evaluate the bioactivity of printed gels, 344SQ lung adenocarcinoma cells were printed in a 3D core/shell architecture. These cells exhibited native phenotypic behavior as evidenced by apparent proliferation and formation of spherical multicellular aggregates. Cells were also printed as pre-formed multicellular aggregates, which appropriately developed invasive protrusions in response to hTGF-β1. Finally, we constructed a simplified model of intratumoral heterogeneity with two separate sub-populations of 344SQ cells, which together grew over 14 days to form a dense regional interface. Together, these studies highlight the potential of multi-material stereolithography to probe heterotypic interactions between distinct cell types in tissue-specific microenvironments. Abbreviations SLA Stereolithography MMSLA Multi-material stereolithography PDMS Poly(dimethyl siloxane) PEG Poly(ethylene glycol) PEGDA Poly(ethylene glycol) diacrylate GelMA Gelatin methacrylate MeHA Methacrylated hyaluronic acid LAP Lithium phenyl-2,4,6-trimethylbenzoylphosphinate kDa Kilodalton 344SQ Murine lung adenocarcinoma cell line hTGF-β1 Human transforming growth factor β1 Mammalian organ function relies on controlled interactions between discrete tissue domains that are physically separate yet biochemically linked. These interfaces are exemplified by vascular capillaries, with which parenchymal and stromal tissues exchange gases, small molecules, and proteins, without transferring larger components such as albumin, fibrinogen, or red and white blood cells. The development and progression of many diseases are often marked by disruptions of these physical boundaries, which regulate the heterotypic dynamics between unique cell populations and extra-cellular components. For example, macular degeneration often begins when retinal blood vessels penetrate through the thin pigment e pithelium1, and cancer metastasis is largely contingent on tumor cell invasion of the surrounding healthy t issues2. To better understand the heterotypic interactions that 1 Department of Bioengineering, Rice University, Houston, TX, USA. 2Department of Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. 3Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. *email: Scientific Reports | (2021) 11:3171 | https://doi.org/10.1038/s41598-021-82102-w 1 Vol.:(0123456789) www.nature.com/scientificreports/ drive these spatial aberrations without the need for costly and variable animal models, researchers must be able to fabricate biological structures with exquisite control over spatial heterogeneity throughout all three dimensions. Sequentially casting distinct combinations of cells and hydrogels into physical molds is the simplest method to achieve spatial heterogeneity. By stacking layers of unique compositions, zonal organization with discrete layers and gradient transitions can be a chieved3. However, there exist tradeoffs between complexity and precision, and these stratified constructs are often limited to uniaxial control. The need for biaxial heterogeneity can be addressed with inkjet p rinting4,5, microcontact p rinting6, or p hotolithography7, but these strategies do not easily scale to full 3D control. Multi-nozzle extrusion printing currently leads the field in heterogeneous b iofabrication8–10, with liquid or shear-thinning bioinks that rapidly solidify as they’re pushed through a circular nozzle, allowing sequential material deposition without risk of material mixing. However, line-by-line deposition of cylindrical filaments can lead to junctional seams and voids that diminish architectural fidelity and may even lead to structural deformation of softer materials during longitudinal cell culture11. Newer approaches within the extrusion framework have used switching Y-junction nozzles to produce monolithic filaments with continuously tunable composition, but the printed structures still suffer from issues of filament interdigitation9. Ultimately, these regional fabrication artifacts may confound interpretations of cellular migration patterns and contact-mediated phenomena, which are often of interest in microphysiologic systems. Compared to multi-nozzle extrusion bioprinting, 3D printing with stereolithography (SLA) does not require physical alignment and fusion of adjacent filaments. Whereas extrusion printers physically deposit cylindrical filaments line-by-line, stereolithography relies on spatially controlled illumination to selectively crosslink liquid bioink into solid hydrogel features. This layer-by-layer printing strategy therefore offers an exciting opportunity to explore multi-material integration with fewer concerns over unintended voids or seams between material domains. SLA also provides access to architectures with over-hanging features such as hollow vessels and freestanding arches12,13, which would otherwise be difficult to achieve with traditional methods of biofabrication. Throughout the SLA printing process, non-illuminated regions remain liquid, leaving behind an excess volume of uncrosslinked bioink that sticks to and coats nascent structures during printing. Therefore, switching the active structure between different materials invariably compromises cellular and biochemical purity. To limit mixing, several strategies have previously been explored. Typically, the active structure is lifted out of the liquid polymer solution and washed with water or saline before returning to the build area. This cleaning step is simple and effective even when performed as a manual process14,15, although automated fluid-exchanging manifolds16,17 and rotating material baths18 have also been developed which help reduce fabrication times. A separate strategy is to clear off residual liquid polymer with (...truncated)