Changes to the cellular microenvironment are an integral characteristic of numerous pathologies, including cancer, fibrosis, and autoimmune disease. Current in vitro methodologies available to study three-dimensional tissue remodeling are ill-suited for high-throughput studies as they are not scalable for large-scale experiments. Combining droplet microfluidics and patterned low-adhesion culture surfaces, we have engineered a workflow to incorporate cell-extracellular matrix (ECM) interactions in a versatile and high-throughput platform that is compatible with existing high-throughput liquid handling systems, enables long-term experiments (>1 month), and is well suited for traditional and novel biological measurements. With our platform, we demonstrate the feasibility of high-throughput ECM remodeling studies with collagen microtissues as one application of a tissue-level function. In this study, we use our workflow to examine ECM remodeling at the tissue, cell, and subcellular levels, leveraging assays ranging from immunohistochemistry and live cell imaging, to proliferation and contraction assays. With our unique culture system, we can track individual constructs over time and evaluate remodeling on several scales for large populations. Finally, we demonstrate the ability to cryopreserve our microtissues while retaining high viability and cell function, an invaluable method that could allow for dissemination and freezing of microtissues after mass production. Using these methods, our ECM-based system becomes a viable platform for modeling diseases characterized by tissue reorganization as well as a scalable method to conduct in vitro cell-based assays for drug screening and high-throughput biological discovery. The described microtissue-microwell workflow is uniquely suited for high-throughput study of extracellular matrix (ECM) remodeling at the molecular, cellular, and tissue levels and demonstrates possibilities of studying progressive, heterogeneous diseases in a way that is meaningful for drug discovery and development. We outline several assays that can be utilized in studying tissue-level diseases and functions that involve cell-ECM interactions and ECM remodeling (e.g., cancer, fibrosis, wound healing) in pursuit of an improved three-dimensional cell culturing system. Finally, we demonstrate the ability to cryopreserve cells encapsulated in microtissue constructs while remaining highly viable, proliferative, and retaining cell functions that are involved in ECM remodeling.
Bibliographical noteFunding Information:
Portions of this work were conducted in the Minnesota Nano Center, which is supported by the National Science Foundation through the National Nanotechnology Coordinated Infrastructure (NNCI) Network under Award Number ECCS-1542202. The authors thank the American Heart Association (13SDG6450000), the National Heart, Lung, and Blood Institute (R21 HL132256), the National Institute of Environmental Health Sciences (R21 ES027622), and the National Science Foundation (CBET 1704332) for financial support. A.L.C. acknowledges the National Science Foundation Graduate Research Fellowship Program (00039202) for support. They also thank Dr. Paolo Provenzano for the use of his multiphoton microscope and donating MDA-MB-231 cells, Dr. Daniel Tschumperlin for donating NHLFs, Dr. Wei Shen for donating NIH 3T3 cells, and Julia Nguyen and Dr. Gregory Vercellotti for isolating and generously donating HUVECs.
© 2019 Mary Ann Liebert, Inc., publishers.
- 3D cell culture
- ECM remodeling
- high-throughput screening