A High-Throughput Workflow to Study Remodeling of Extracellular Matrix-Based Microtissues

Abstract

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.

Stromal architecture directs early dissemination in pancreatic ductal adenocarcinoma

“…Matrigel microtissues approximately 150-200μm in diameter were fabricated modifying previously established protocols ( 60, 61 ). Matrigel (Corning) was thawed on ice overnight and diluted to a concentration of 6 mg/mL with Dulbecco's phosphate-buffered saline (DPBS).…”

Cryopreservation as a Key Element in the Successful Delivery of Cell-Based Therapies—A Review

“…Cell encapsulation may provide greater functional performance, but spheroids are also difficult to cryopreserve due to physical ice damage suffered by the spheroid and the protracted cell dehydration times necessary during cooling to accommodate the diffusion distances within the spheroids ( 59 , 65 ). Larger aggregates of cells, perhaps included in alginate beads, complicate cryopreservation as varying diffusion distances, thermal gradients and limiting membrane permeabilities can be present, and this complexity increases when the size of candidate product is increased e.g., cells growing around a scaffold, either natural or artificial ( 142 , 143 ). Cryopreservation of cells or spheroids attached to scaffolds or other types of 3D constructs to support cell proliferation for tissue engineering must consider both damage to the scaffold itself as well as cell detachment caused by contraction and expansion of the scaffold material during cooling and warming ( 144 – 146 ).…”

Microscale Collagen and Fibroblast Interactions Enhance Primary Human Hepatocyte Functions in 3-Dimensional Models

“…PDMS stamps with ~1000 micropillars were placed into the molten agarose within each well of a 24-well polystyrene plate. After the agarose cooled, the PDMS stencils were removed, forming ~300 µm diameter x ~300 µm deep wells (microwells) in the casted agarose 16 . PDMS devices and agarose microwells were sterilized via autoclaving or 70% ethanol treatment (1 hour), respectively.…”

“…However, large (>500 µm) ECM hydrogels that encapsulate cells pose significant diffusion limitations for oxygen and nutrients in the construct's core in the absence of a functional vasculature 14 ; this limitation can be mitigated by miniaturizing the hydrogel scaffolds to ~100-300 µm, albeit it is not trivial to create reproducible hydrogel scaffolds of this size via manual pipetting. Thus, in recent years, investigators have employed microfluidics to produce highly monodisperse microscale emulsions (droplets) in a highthroughput format 15,16 . This so-called 'droplet microfluidics' is ideally suited to precisely tune at the microscale cell-cell and cell-ECM interactions and determine optimal conditions for cell survival and function.…”

“…While the studies above show the utility of droplet microfluidics for the high-throughput generation of functional hepatocyte/fibroblast droplets using hepatic cell lines and primary rat hepatocytes, the significant differences between these cell types and PHHs in liver functions and drug outcomes necessitates studies with PHHs to determine optimal microenvironmental cues for their long-term culture 20,21 . Therefore, here we sought to adapt our previously developed droplet microfluidic device useful for rapidly fabricating protein microgels 15,16 to a) determine the optimal device and culture conditions for the creation of PHH/collagen droplets (microtissues) in a multiwell plate format, b) elucidate the role of different fibroblast populations (3T3-J2 murine embryonic fibroblasts and primary human hepatic stellate cells) on the long-term (up to 6 weeks) enhancement and stabilization of PHH functions when the two cell types were either co-encapsulated within the collagen droplets or when the fibroblasts were 'coated' on the outside of the PHH-containing collagen droplets, c) determine the differences in hepatic functional output within microtissues relative to conventional self-assembled spheroids and collagen bulk hydrogels (macrogels), and d) determine the utility of the microtissues for assays in drug development, specifically drug-mediated CYP450 induction and drug-induced hepatotoxicity.…”