Understanding Cell Behavior in Precise Multifunctional Biomimetic Matrices Prepared via 3D Printing

/ABSTRACT The lack of cell culture strategies that faithfully replicate both form and function of the extracellular matrix (ECM) hinders our ability to effectively monitor, diagnose, and treat disease. This arises from the contemporary reliance on in vitro cell culture methods that fail to replicate complex biological structures, while in vivo cell cultures use animal models that have disparate physiology from humans. As a result, there is a gap in fundamental knowledge surrounding how cells respond to various stimuli (or cues), limiting our ability to understand and fight illness. Filling this gap necessitates new precision processes to create 3D structures that emulate the natural ECM for in vitro cell culture. Herein, a feedback loop between digital light processing (DLP) 3D printing and cell studies will provide new process-structure-function relationships between the technology to create biomimetic scaffolds and concomitant cell-matrix interactions. Open fundamental questions to be addressed herein include: 1) To what extent does using visible and near infrared light in DLP 3D printing improve cell viability and metabolic activity over using UV light? 2) What are the optimal microscopic mesh sizes and macroscopic pore geometries to facilitate uniform cell morphology and proliferation? 3) How does the layered topography of DLP 3D printed structures influence cell behavior (e.g., adhesion, mobility, and differentiation)? 4) How do cells respond to sharp vs. smooth interfaces where a cue is transitioning (e.g., stiffness gradients)? 5) How do cellular responses vary for individual vs. combined mechanical and biochemical cues? Answers to these questions will be accomplished using human mesenchymal stem cells (hMSCs) as a model system given their multipotent versatility and microenvironment sensitivity that is representative of many cell types. DLP 3D printing was selected as the fabrication platform owing to its unparalleled combination of speed, resolution, and low cost. However, this technique to-date has been predominantly restricted to harmful UV light exposure, toxic acrylic resins, and the production of rigid homogeneous parts that differ from the vital heterogeneity and softness present in many ECMs. The Page lab is uniquely positioned to address these issues given their existing expertise and infrastructure that will enable integration of heterogeneity into biomimetic structures by using benign visible to near infrared (multi-)color and intensity (grayscale) light projection. This will allow for precise 3D spatial control over network structure, mechanical properties, and protein tagging. hMSCs will be directly encapsulated in synthetic ECM mimetics that emulate tissues ranging in stiffness from lungs (“supersoft”) to ligaments (“hard”). Additionally, localization of proteins (e.g., growth factors) will be used to direct adhesion, movement, and growth of hMSCs. Cell viability and behavior within the 3D scaffolds will be systematically characterized to elicit structure-function relationships that address the above fundamental questions and that will inform further bioprinting optimization and cell culture. If successful, this work will provide a platform to improve our capability to monitor, diagnose, and treat diseases in a non-invasive in vitro manner. Project Number: 1R35GM156809-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Zachariah Page | Institution: UNIVERSITY OF TEXAS AT AUSTIN, AUSTIN, TX | Award Amount: $385,671 | Activity Code: R35 | Study Section: Special Emphasis Panel[ZRG1 MBBC-F (55)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11019379