The potential of liquid marbles for biomedical applications: a critical review

Liquid marbles (LM) are freestanding droplets covered by micro/nanoparti- cles with hydrophobic/hydrophilic properties, which can be manipulated as a soft solid. The phenomenon that generates these soft structures is regarded as a different method to generate a superhydrophobic behavior in the liquid/ solid interface without modifying the surface. Several applications for the LM have been reported in very different fields, however the developments for bio- medical applications are very recent. At first, the LM properties are reviewed, namely shell structure, LM shape, evaporation, floatability and robustness. The different strategies for LM manipulation are also described, which make use of magnetic, electrostatic and gravitational forces, ultraviolet and infrared radiation, and approaches that induce LM self-propulsion. Then, very distinc- tive applications for LM in the biomedical field are presented, namely for diagnostic assays, cell culture, drug screening and cryopreservation of mam- malian cells. Finally, a critical outlook about the unexplored potential of LM for biomedical applications is presented, suggesting possible advances on this emergent scientific area. The authors acknowledge funding from the European Research Council grant agreement ERC-2012-ADG 20120216-321266 for project ComplexiTE. N. M. Oliveira acknowledges the financial support from Portuguese Foundation for Science and Technology - FCT (Grant SFRH/BD/73172/2010), from the financial program POPH/FSE from QREN.info:eu-repo/semantics/publishedVersio

Multizone Paper Platform for 3D Cell Cultures

In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other environmental factors (temperature, mechanical stress, etc). The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously (“cells-in-gels-in-paper” or CiGiP), this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM) gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers) containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D culture, by peeling apart the sheets of paper, “sections” all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures

Oxygen-Controlled Three-Dimensional Cultures to Analyze Tumor Angiogenesis

Tumor angiogenesis is controlled by the integrated action of physicochemical and biological cues; however, the individual contributions of these cues are not well understood. We have designed alginate-based microscale tumor models to define the distinct importance of oxygen concentration, culture dimensionality, and cell–extracellular matrix interactions on the angiogenic capability of oral squamous cell carcinoma, and have verified the relevance of our findings with U87 glioblastoma cells. Our results revealed qualitative differences in the microenvironmental regulation of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) secretion in three-dimensional (3D) culture. Specifically, IL-8 secretion was highest under ambient conditions, whereas VEGF secretion was highest in hypoxic cultures. Additionally, 3D integrin engagement by RGD-modified alginate matrices increased IL-8 secretion independently of oxygen, whereas VEGF secretion was only moderately affected by cell–extracellular matrix interactions. Using two-dimensional migration assays and a new 3D tumor angiogenesis model, we demonstrated that the resulting angiogenic signaling promotes tumor angiogenesis by increasing endothelial cell migration and invasion. Collectively, tissue-engineered tumor models improve our understanding of tumor angiogenesis, which may ultimately advance anticancer therapies