2024 AIChE Annual Meeting

(413h) A High Throughput, Versatile Approach for Probing Macrophage Responses to Microenvironment Cues in 3D Bioprinted Synthetic Extracellular Matrices.

Authors

Graf, J. - Presenter, Lafayette College
Moore, D., University of Delaware
Fromen, C., University of Delaware
Kloxin, A., University of Delaware
Grimes, C., University of Delaware
Macrophages are key innate immune cells that serve as the first line of defense against foreign pathogens and particulates through phagocytosis and proinflammatory, anti-inflammatory, or anti-microbial signaling. While two-dimensional (2D) culture on tissue-culture plastic remains the standard of practice, the field is shifting toward compliant three-dimensional (3D) culture models for testing hypotheses about cell-microenvironment interactions and providing well-defined, tunable, and robust systems that better mimic aspects of the native extracellular matrix (ECM). However, the low throughput of many manually-prepared 3D culture models presents challenges for translation of assays and their broad and accessible use. Bioprinting has the potential to be a consistent and high-throughput method for generating synthetic ECMs in a multi-well plate format for studying cellular interactions and treatment strategies within more physiologically relevant microenvironments. For achieving this, we have established pertinent workflows and demonstrate the relevance of this approach for a model human monocyte cell line (THP-1) that can be differentiated into macrophages, with insights into macrophage responses to different stimuli (e.g., stimulation with biochemical and biophysical cues, pathogens, applied treatments). Our data further support the utility of this innovative methodology and technology for culturing a variety of macrophage types, including primary bone-marrow derived macrophages.

Hydrogels were created with the RASTRUMTM bioprinter, using PEG- and peptide-based bioinks that react using thiol-maleimide Michael addition chemistry. Inks incorporated integrin-binding peptides RGD, GFOGER, and YIGSR, inspired by the ECM proteins of native human tissues. Human THP-1 macrophages were encapsulated within bioprinted hydrogel-based synthetic ECMs and differentiated into macrophages. Macrophage viability, morphology, phenotype, and inflammatory response to stimuli were assessed within synthetic ECMs with stiffnesses relevant to healthy (storage modulus (G’)~0.7 and 1.1 kPa) and fibrotic (G’~3.0 and 4.8 kPa) lung tissues. Hydrogels were shown to support macrophage viability, and flow cytometry and ELISA revealed appropriate macrophage polarization and cytokine secretion in response to microenvironmental stimuli. We then applied our model 3D cultures to study immune response to invasion of a bacterial pathogen implicated in hospital born lung infections and mortality.

This work represents a platform for modeling and understanding immune response in physiologically relevant tissue microenvironments, with opportunities for well-defined, yet more complex co-cultures. Future directions aim to further establish healthy and diseased tissue models to understand how immune cells respond to pathogens, particulates, treatments, and other stimuli relevant to chronic lung diseases.