(513a) Control of Synthetic ECM Context to Direct Cell Morphology and Cell Adhesion in 2D and 3D | AIChE

(513a) Control of Synthetic ECM Context to Direct Cell Morphology and Cell Adhesion in 2D and 3D


Tibbitt, M. W. - Presenter, University of Colorado, Howard Hughes Medical Institute
Anseth, K. S. - Presenter, University of Colorado-Boulder
Kloxin, A. M. - Presenter, University of Delaware
Dyamenahalli, K. U. - Presenter, University of Colorado

Cell-extracellular matrix (ECM) interactions are known to affect cell morphology and cytoskeletal organization, which direct critical cell functions such as proliferation, differentiation, and migration.1 To investigate how cell-ECM interactions direct cellular processes, progenitor cells have been cultured on 2D and within 3D defined scaffolds with varied elasticities, geometries, and adhesive contexts.2,3 This seminal work has led to a nascent understanding of cellular mechanotransdution; however, the bulk of these experiments are conducted with static scaffolds that fail to recapitulate the dynamic nature of the ECM and fail to capture how changes in the ECM are mechanotransduced to influence cell function. Here, we present a unique experimental strategy in which a photolabile poly(ethylene glycol) (PEG)-based hydrogel is used to dynamically tune the ECM with light. This photomodulation is cytocompatible, so dynamic changes in material properties can be achieved in the presence of cells with precise and predictable control. Our aim is to exploit this photolabile synthetic ECM mimic as a platform where user-defined, dynamic cell-ECM interactions can be studied in vitro and build a fundamental understanding of active mechanotransduction. The specific platform employed is a photodegradable hydrogel with an entrapped adhesion protein, fibronectin, that facilitates cell adhesion. The gel is formed by free-radical co-polymerization of a photolabile PEG diacrylate crosslinker with PEG monoacrylate.4 Polymerization and gelation are conducted in the presence of human mesenchymal stem cells (hMSCs) with greater than 90% viability. Subsequent irradiation (365nm single photon or 740nm two-photon) is employed to tune the elasticity (polymer density) or adhesive context of the gel in the presence of cells in both 2D and 3D. Specifically, gradients in polymer density through the z-dimension of photolabile hydrogels were introduced with 10 mW/cm2, 365nm flood irradiation to investigate how hMSCs respond to changes in network density in 3D.5 hMSC spreading was observed preferentially in regions of decreased polymer density, and gradients were used to control cell morphology spatially in 3D. hMSCs were also transfected with a GFP-actin plasmid to visualize cytoskeletal organization and seeded on the surface of photolabile hydrogels. Femtosecond pulsed, two-photon irradiation (740nm, 1.25nJ/pulse) was used to selectively erode the photolabile hydrogel at the cell-gel interface, removing defined cell adhesion regions with micron-scale resolution.6 In this manner, we precisely and predictably modified the adhesive context of individual hMSCs and monitored each cell's morphological and cytoskeletal response. Disruption of adhesive regions induced subcellular detachment and the detachment dynamics were quantified, indicating that subcellular detachment occurs more slowly from soft hydrogel substrates than stiffer glassy substrates. Ultimately, this material platform provides a unique system for cell culture, affording user-defined control of the 2D and 3D ECM density, geometry, and adhesive context at any point in space and time.


1. Tibbitt MW, Anseth KS, Biotech Bioeng 103 (2009) 655-663

2. Engler AJ, Sen S, Sweeney HL, Discher DE Cell 126 (2006) 677-689

3. Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE Science 276 (1997) 1425-1428

4. Kloxin AM, Kasko AM, Salinas CN, Anseth KS Science 324 (2009) 59-63

5. Kloxin AM, Tibbitt MW, Kasko AM, Fairbairn JA, Anseth KS Adv Matls 22 (2010) 61-66

6. Tibbitt MW, Kloxin AM, Dyamenahalli KU, Anseth KS Soft Matter Submitted