Spatially controlled photo-patterning of multi-material bioactive hydrogels

Abstract

Event Abstract Back to Event Spatially controlled photo-patterning of multi-material bioactive hydrogels Bagrat Grigoryan1*, Jonathon D. Roybal2, Paul T. Greenfield1, Alexander J. Zaita1, Samantha J. Paulsen1, Jacob L. Albritton1, Anderson Ta1, Don L. Gibbons2, 3 and Jordan S. Miller1 1 Rice University, Bioengineering, United States 2 The University of Texas M.D. Anderson Cancer Center, Thoracic/Head and Neck Medical Oncology, United States 3 The University of Texas, MD Anderson Cancer Center, Molecular and Cellular Oncology, United States Introduction: While fabrication of simple microenvironments to study cell fate and function has been achieved using soft lithography and photolithography techniques, mimicking the complex, heterogeneous patterns of native tissue in vitro remains challenging due to lack of available simple, automated patterning technologies[1]. To this end, we developed a stereolithography (SLA)-based 3D printer for automated fabrication of hydrogels containing microstructures composed of multi-materials for applications towards understanding the lung tumor microenvironment. Materials and Methods: We developed and characterized our custom SLA-based 3D printer to spatially control the patterning of multiple materials within a single layer and across successive layers. We performed photorheological characterization of prepolymer formulations containing poly(ethylene glycol) diacrylate (PEGDA) and lithium acylphosphinate (LAP) photoinitiator to quantitatively understand the gelation kinetics and modulus of a single layer of hydrogel. Patterned entrapped fluorophore molecules facilitated characterization of the fidelity of multi-material patterns. To demonstrate biocompatibility of our technology, we added hMSCs into the prepolymer solution and performed LIVE/DEAD staining of printed cell-laden hydrogels at various time points. We further demonstrate the biocompatibility of our technology by adding a murine metastatic lung adenocarcinoma cell line, 344SQ, into the prepolymer solution to fabricate bioactive PEG-based gels containing cell-adhesive CGRGDS ligand and MMP-sensitive peptides incorporated into the polymer backbone[2]. We investigated cell viability and function by monitoring the innate ability of 344SQ cells to form into aggregates and lumenize over time in the proper microenvironment. Results and Discussion: The photorheological assay provided us with information on the exposure duration needed for a certain layer thickness as a function of PEG molecular weight, PEG concentration, and LAP concentration in the prepolymer formulation for reproducible fabrication of hydrogels (Figure 1A). Fluorescent microscopy images demonstrated fabrication of features as small as 300µm in diameter as well as precise spatial control in photo-patterning of multiple materials within the same plane (Figure 1B). Encapsulation of cells in bioactive PEG-based gels demonstrated cell viability greater than 90% after 2 days of culture (Figure 1C-Top). Encapsulated 344SQ cells retained their biological activity by forming into aggregates and lumenizing throughout the 2-week long culture (Figure 1C-Bottom). Conclusion: We have developed an open-source SLA-based 3D printer that enables us to automatically fabricate hydrogels containing bioactive microstructures with spatial control of multiple materials at a medium-high throughput. We utilized this technology to encapsulate hMSCs and metastatic cells in PEG-based hydrogels with high cell viability and function. We believe the automated nature of our developed system will enable researchers to recapitulate intricate microenvironments for studying complex cellular interactions in vitro without facing difficulties associated with precisely aligning masks with traditional photolithographic techniques. Thanks to the Open Source Hardware Association, RepRap.org, and related projects and companies that support worldwide, open standardization of 3D printing. This work was supported in part by NSF Graduate Research Fellowship (BG).