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IntroductionCell structure and migration is impacted by the mechanical properties and geometry of the cell adhesive environment. Most studies to date investigating the effects of 3D environments on cells have not controlled geometry at the single-cell level, making it difficult to understand the influence of 3D environmental cues on single cells. Here, we developed microwell platforms to investigate the effects of 2D vs. 3D geometries on single-cell F-actin and nuclear organization.MethodsWe used microfabrication techniques to fabricate three polyacrylamide platforms: 3D microwells with a 3D adhesive environment (3D/3D), 3D microwells with 2D adhesive areas at the bottom only (3D/2D), and flat 2D gels with 2D patterned adhesive areas (2D/2D). We measured geometric swelling and Young's modulus of the platforms. We then cultured C2C12 myoblasts on each platform and evaluated the effects of the engineered microenvironments on F-actin structure and nuclear shape.ResultsWe tuned the mechanical characteristics of the microfabricated platforms by manipulating the gel formulation. Crosslinker ratio strongly influenced geometric swelling whereas total polymer content primarily affected Young's modulus. When comparing cells in these platforms, we found significant effects on F-actin and nuclear structures. Our analysis showed that a 3D/3D environment was necessary to increase actin and nuclear height. A 3D/2D environment was sufficient to increase actin alignment and nuclear aspect ratio compared to a 2D/2D environment.ConclusionsUsing our novel polyacrylamide platforms, we were able to decouple the effects of 3D confinement and adhesive environment, finding that both influenced actin and nuclear structure.

Human-induced pluripotent stem cell-derived cardiomyocytes are a potentially unlimited cell source and promising patient-specific in vitro model of cardiac diseases. Yet, these cells are limited by immaturity and population heterogeneity. Current in vitro studies aiming at better understanding of the mechanical and chemical cues in the microenvironment that drive cellular maturation involve deformable materials and precise manipulation of the microenvironment with, for example, micropatterns. Such microenvironment manipulation most often involves microfabrication protocols which are time-consuming, require cleanroom facilities and photolithography expertise. Here, we present a method to increase the scale of the fabrication pipeline, thereby enabling large-batch generation of shelf-stable microenvironment protein templates on glass chips. This decreases fabrication time and allows for more flexibility in the subsequent steps, for example, in tuning the material properties and the selection of extracellular matrix or cell proteins. Further, the fabrication of deformable hydrogels has been optimized for compatibility with these templates, in addition to the templates being able to be used to acquire protein patterns directly on the glass chips. With our approach, we have successfully controlled the shapes of cardiomyocytes seeded on Matrigel-patterned hydrogels.

Introduction Cell structure and migration is impacted by the mechanical properties and geometry of the cell adhesive environment. Most studies to date investigating the effects of 3D environments on cells have not controlled geometry at the single-cell level, making it difficult to understand the influence of 3D environmental cues on single cells. Here, we developed microwell platforms to investigate the effects of 2D vs 3D geometries on single-cell F-actin and nuclear organization. Methods We used microfabrication techniques to fabricate three polyacrylamide platforms: 3D microwells with a 3D adhesive environment ( 3D/3D ), 3D microwells with 2D adhesive areas at the bottom only ( 3D/2D ), and flat 2D gels with 2D patterned adhesive areas ( 2D/2D ). We measured geometric swelling and Young’s modulus of the platforms. We then cultured C2C12 myoblasts on each platform and evaluated the effects of the engineered microenvironments on F-actin structure and nuclear shape. Results We tuned the mechanical characteristics of the microfabricated platforms by manipulating the gel formulation. Crosslinker ratio strongly influenced geometric swelling whereas total polymer content primarily affected Young’s modulus. When comparing cells in these platforms, we found significant effects on F-actin and nuclear structures. Our analysis showed that a 3D/3D environment was necessary to increase actin and nuclear height. A 3D/2D environment was sufficient to increase actin alignment and nuclear aspect ratio compared to a 2D/2D environment. Conclusions Using our novel polyacrylamide platforms, we were able to decouple the effects of 3D confinement and adhesive environment, finding that both influenced actin and nuclear structure.

While performing several functions, adherent cells deform their surrounding substrate via stable adhesions that connect the intracellular cytoskeleton to the extracellular matrix. The traction forces that deform the substrate are studied in mechanotrasduction because they are affected by the mechanics of the extracellular milieu. We review the development and application of two methods widely used to measure traction forces generated by cells on 2D substrates: (i) traction force microscopy with polyacrylamide hydrogels and (ii) calculation of traction forces with arrays of deformable microposts. Measuring forces with these methods relies on measuring substrate displacements and converting them into forces. We describe approaches to determine force from displacements and elaborate on the necessary experimental conditions for this type of analysis. We emphasize device fabrication, mechanical calibration of substrates and covalent attachment of extracellular matrix proteins to substrates as key features in the design of experiments to measure cell traction forces with polyacrylamide hydrogels or microposts. We also report the challenges and achievements in integrating these methods with platforms for the mechanical stimulation of adherent cells. The approaches described here will enable new studies to understand cell mechanical outputs as a function of mechanical inputs and advance the understanding of mechanotransduction mechanisms.

While performing several functions, adherent cells deform their surrounding substrate via stable adhesions that connect the intracellular cytoskeleton to the extracellular matrix. The traction forces that deform the substrate are studied in mechanotrasduction because they are affected by the mechanics of the extracellular milieu. We review the development and application of two methods widely used to measure traction forces generated by cells on 2D substrates: (i) traction force microscopy with polyacrylamide hydrogels and (ii) calculation of traction forces with arrays of deformable microposts. Measuring forces with these methods relies on measuring substrate displacements and converting them into forces. We describe approaches to determine force from displacements and elaborate on the necessary experimental conditions for this type of analysis. We emphasize device fabrication, mechanical calibration of substrates and covalent attachment of extracellular matrix proteins to substrates as key features in the design of experiments to measure cell traction forces with polyacrylamide hydrogels or microposts. We also report the challenges and achievements in integrating these methods with platforms for the mechanical stimulation of adherent cells. The approaches described here will enable new studies to understand cell mechanical outputs as a function of mechanical inputs and advance the understanding of mechanotransduction mechanisms.