Here we demonstrate an efficient method to fabricate large-domain monodisperse foam scaffolds made of gelatin for 3D cell culture. We tested three distinct tissue cell types cultured in foam scaffolds composed of uniform spherical pores. The cells displayed appropriate morphological and physiological characteristics: epithelial cells formed cyst-like structures and were polarized inside pores, myoblasts adopted a tubular structure and fused into myotubes, and fibroblasts exhibited a wide variety of morphologies. Scaffolds with uniform pores can thus provide a platform for systematic study of 3D cell-matrix interactions.
Neonatal ventricular myocytes (CM) have long been used as an in vitro model for hypertrophy studies. In conventional 2D culture, CM lack axial orientation and rhythmic electrical stimulation. Micropatterned cultures can restrict cell attachment to narrow stripes, leading to enhanced axial orientation, particularly in spontaneously contracting CM (Rohr et. al., Circ Res, 1991). In this study, we investigated the effect of continous electrical field stimulation (CES) on micropatterned CM and examined their response to hypertrophic stimulation. Rat CM plated in serum-containing media selectively attached to stripes (100 mm x 10 mm) of fibronectin (FN) that were microcontact-printed onto coverslips. CM cultures were subjected to CES (1 Hz, 5 V/cm) for 48 hrs, with the current applied parallel or perpendicular to FN stripes. To induce a hypertrophic response, micropatterned CM were incubated for 48 hrs in serum-free medium with the a 1 adrenoceptor agonist phenylephrine (PE, together with timolol). We determined that the size, minor/major axis ratio and angles relative to FN stripes of DAPI-stained nuclei can be used as surrogate measures of CM size, elongation and alignment, respectively. Compared to unpaced CM, parallel CES increased nuclear size (10285121 vs. 798587 mm 2 , P<0.001), elongation (minor/major axis: 0.7650.10 vs. 0.8450.08, P<0.001) and alignment (P<0.001, Mardia-Watson-Wheeler circular statistics). Perpendicular CES caused similar but significantly less pronounced changes. PE stimulation increased nuclear size (809593 vs. 682599 mm 2 , P<0.05), but did not increase elongation or alignment with or without CES. In conclusion, CES can be used to enhance the degree of differentiation of micropatterned CM due to continuous electrical activation and/or contractions and does not interfere with their hypertrophic response. Continuously paced micropatterned CM represent an advanced model for the investigation of hypertrophic responses and mechanisms and may be suitable for other applications.
To monitor cellular processes in individual cells, it is important to measure the concentrations of intracellular metabolites and to retrieve them for analysis. The use of functionalized polyelectrolyte microcapsules as intracellular sensors for in vivo reporting is persented. Capsules loaded with streptavidinrhodamine, which was introduced into fibroblasts by electroporation, autonomously escaped from an endocytic compartment and effi ciently recruited biotin-fluorescein from the cytosol. This work demonstrates the utility of polyelectrolyte microcapsules for intracellular capture of metabolites and eventually for drug delivery on an organismic level.
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