Speaker
Description
Introduction
Three-dimensional (3D) microtissue spheroids replicate native-like microenvironments and thus provide a model platform to study cell-matrix dynamics. Furthermore, modular bioassembly of these cell-dense units allows for precise control over 3D tissue architecture, while maintaining physiological cellular microenvironments. However, successful biofabrication of large-scale tissues from modular microtissue units requires microscale fusion events involving cell migration and cohesive extracellular matrix (ECM) integration [1]. Therefore, understanding interactions between tissue modules is essential for advancing scalable human tissues. As such, metabolic labelling of nascent ECM formation is a promising approach to explore the spatiotemporal dynamics of ECM development. Here, non-canonical amino acids containing biorthogonal functional groups are incorporated into newly secreted proteins and fluorescently labelled to visualize nascent matrix deposition (Figure 1A). In this study, we applied a click chemistry-based approach to metabolically label nascent proteins in 3D cartilage microtissues to investigate the spatial and temporal development of tissue growth and matrix deposition across microtissues and during tissue fusion events.
Methods
High-density human articular chondrocyte microtissues (0.25x106 cells/microtissue) were formed using high-throughput centrifugation in a V-bottom plate and allowed to mature over 14 days. Next, microtissues were bioassembled into 3D-printed, polycaprolactone (PCL) scaffolds and cultured for 14 days to facilitate tissue fusion [2, 3]. During spheroid formation and fusion, microtissues were treated with a non-canonical amino acid, L-azidohomoalanine (AHA; 100 µM), for 1 day at early (Day 1) or late (Day 14) time points. AHA-incorporated microtissues were fluorescently labelled with a dibenzocyclooctyne (DBCO) functionalized fluorophore to visualize nascent ECM formation occurring only during AHA treatment (Figure 1A) [4]. Spatiotemporal nascent matrix deposition was quantified and analysed qualitatively (safranin‑O, collagen I/II staining) across microtissues and in fusion regions.
Results
Staining for nascent proteins revealed widespread protein deposition across microtissues, with a 300 % increase in staining intensity at the periphery, suggesting enhanced protein secretion due to greater cellular activity at the microtissue surface. Comparing the protein deposition over time, we observed minimal ECM deposition during the first 24 hours of microtissue formation, followed by a 6.4-fold increase after 14 days of culture, determined by fluorescence intensity quantification (Figure 1B). Application of metabolic labelling during microtissue fusion within the 3D bioassembly model enabled evaluation of nascent matrix formation at the fusion interface, leading a pathway to probe ECM formation across larger constructs (Figure 1C).
Discussion
We present an innovative metabolic labelling approach to study spatiotemporal matrix evolution in complex, multicellular 3D tissues, revealing mechanisms involved in tissue formation, maturation and fusion. Our findings highlight spatial differences and temporal dynamics of ECM deposition across 3D microtissues. Understanding ECM dynamics during tissue fusion is crucial for engineering large-scale 3D constructs. Our fusion model offers a platform to design, evaluate and optimize next-generation tissue spheroid modules and their interaction for scaled-up biofabrication, supporting the development of functional regenerative therapies.
References
[1] Wolf, K. et al., Cell Stem Cell, 29(5), 2022
[2] Lindberg G. et al., Adv Sci, 8(22), 2021
[3] Veenendaal L. et al., Adv Mater Int, 9(31), 2022
[4] Loebel, C. et al., Nat mater, 18(8), 2019
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