Dataset Information

Multiwell Combinatorial Hydrogel Array for High-Throughput Analysis of Cell-ECM Interactions.

ABSTRACT: Biophysical cues in the extracellular matrix (ECM) regulate cell behavior in a complex, nonlinear, and interdependent manner. To quantify these important regulatory relationships and gain a comprehensive understanding of mechanotransduction, there is a need for high-throughput matrix platforms that enable parallel culture and analysis of cells in various matrix conditions. Here we describe a multiwell hyaluronic acid (HA) platform in which cells are cultured on combinatorial arrays of hydrogels spanning a range of elasticities and adhesivities. Our strategy utilizes orthogonal photopatterning of stiffness and adhesivity gradients, with the stiffness gradient implemented by a programmable light illumination system. The resulting platform allows individual treatment and analysis of each matrix environment while eliminating contributions of haptotaxis and durotaxis. In human mesenchymal stem cells, our platform recapitulates expected relationships between matrix stiffness, adhesivity, and cell mechanosensing. We further applied the platform to show that as integrin ligand density falls, cell adhesion and migration depend more strongly on CD44-mediated interactions with the HA backbone. We anticipate that our system could bear great value for mechanistic discovery and screening where matrix mechanics and adhesivity are expected to influence phenotype.

SUBMITTER: Lei R

PROVIDER: S-EPMC8900525 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Multiwell Combinatorial Hydrogel Array for High-Throughput Analysis of Cell-ECM Interactions.

Lei Ruoxing R Akins Erin A EA Wong Kelly C Y KCY Repina Nicole A NA Wolf Kayla J KJ Dempsey Garrett E GE Schaffer David V DV Stahl Andreas A Kumar Sanjay S

ACS biomaterials science & engineering 20210524 6

Biophysical cues in the extracellular matrix (ECM) regulate cell behavior in a complex, nonlinear, and interdependent manner. To quantify these important regulatory relationships and gain a comprehensive understanding of mechanotransduction, there is a need for high-throughput matrix platforms that enable parallel culture and analysis of cells in various matrix conditions. Here we describe a multiwell hyaluronic acid (HA) platform in which cells are cultured on combinatorial arrays of hydrogels ...[more]

PMID: 34028263

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Project description:BackgroundMiniaturization of biochemical reaction volumes within artificial microcompartments has been the key driver for directed evolution of several catalysts in the past two decades. Typically, single cells are co-compartmentalized within water-in-oil emulsion droplets with a fluorogenic substrate whose conversion allows identification of catalysts with improved performance. However, emulsion droplet-based technologies prevent cell proliferation to high density and preclude the feasibility of biochemical reactions that require the exchange of small molecule substrates. Here, we report on the development of a high-throughput screening method that addresses these shortcomings and that relies on a novel selective permeable polymer hydrogel microcapsule.ResultsHollow-core polyelectrolyte-coated chitosan alginate microcapsules (HC-PCAMs) with selective permeability were successfully constructed by jet break-up and layer-by-layer (LBL) technology. We showed that HC-PCAMs serve as miniaturized vessels for single cell encapsulation, enabling cell growth to high density and cell lysis to generate monoclonal cell lysate compartments suitable for high-throughput analysis using a large particle sorter (COPAS). The feasibility of using HC-PCAMs as reaction compartments which exchange small molecule substrates was demonstrated using the transpeptidation reaction catalyzed by the bond-forming enzyme sortase F from P. acnes. The polyelectrolyte shell surrounding microcapsules allowed a fluorescently labelled peptide substrate to enter the microcapsule and take part in the transpeptidation reaction catalyzed by the intracellularly expressed sortase enzyme retained within the capsule upon cell lysis. The specific retention of fluorescent transpeptidation products inside microcapsules enabled the sortase activity to be linked with a fluorescent readout and allowed clear separation of microcapsules expressing the wild type SrtF from those expressing the inactive variant.ConclusionA novel polymer hydrogel microcapsule-based method, which allows for high-throughput analysis based on encapsulation of single cells has been developed. The method has been validated for the transpeptidation activity of sortase enzymes and represents a powerful tool for screening of libraries of sortases, other bond-forming enzymes, as well as of binding affinities in directed evolution experiments. Moreover, selective permeable microcapsules encapsulating microcolonies provide a new and efficient means for preparing novel caged biocatalyst and biosensor agents.

Dataset Information

Multiwell Combinatorial Hydrogel Array for High-Throughput Analysis of Cell-ECM Interactions.

Publications

Multiwell Combinatorial Hydrogel Array for High-Throughput Analysis of Cell-ECM Interactions.

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