Grow with the Flow:When Morphogenesis Meets Microfluidics

Developmental biology has advanced the understanding of the intricate and dynamic processes involved in the formation of an organism from a single cell. However, many gaps remain in the knowledge of embryonic development, especially regarding tissue morphogenesis. A possible approach to mimic such phenomena uses pluripotent stem cells in in vitro morphogenetic models. Herein, these systems are summarized with emphasis on the ability to better manipulate and control cellular interfaces with either liquid or solid materials using microengineered tools, which is critical for attaining deeper insights into pattern formation and stem cell differentiation during organogenesis. The role of conventional and customized cell-culture systems in supporting important advances in the field of morphogenesis is discussed, and the fascinating role that material sciences and microengineering currently play and are expected to play in the future is highlighted. In conclusion, it is proffered that continued microfluidics innovations when applied to morphogenesis promise to provide important insights to advance many multidisciplinary fields, including regenerative medicine

A New Microengineered Platform for 4D Tracking of Single Cells in a Stem-Cell-Based In Vitro Morphogenesis Model

Recently developed stem-cell-based in vitro models of morphogenesis can help shed light on the mechanisms involved in embryonic patterning. These models are showcased using traditional cell culture platforms and materials, which allow limited control over the biological system and usually do not support high-content imaging. In contrast, using advanced microengineered tools can help in microscale control, long-term culture, and real-time data acquisition from such biological models and aid in elucidating the underlying mechanisms. Here, a new culturing, manipulation and analysis platform is described to study in vitro morphogenesis using thin polycarbonate film-based microdevices. A pipeline consisting of open-source software to quantify 3D cell movement using 4D image acquisition is developed to analyze cell migration within the multicellular clusters. It is shown that the platform can be used to control and study morphogenesis in non-adherent cultures of the P19C5 mouse stem cell line and mouse embryonic stem cells (mESCs) that show symmetry breaking and axial elongation events similar to early embryonic development. Using the new platform, it is found that localized cell proliferation and coordinated cell migration result in elongation morphogenesis of the P19C5 aggregates. Further, it is found that polarization and elongation of mESC aggregates are dependent on directed cell migration

Polystyrene Pocket Lithography - Sculpting Plastic with Light

Tissue culture ware polystyrene is the gold standard for in vitro cell culture. While microengineering techniques can create advanced cell microenvironments in polystyrene, they require specialized equipment and reagents, which hinder their accessibility for most biological researchers. We developed and validated an economical and easily accessible method for fabricating microstructures directly in polystyrene with sizes approaching subcellular dimensions while requiring minimal processing time. The process involves deep ultraviolet irradiation through a shadow mask or ink pattern using inexpensive, handheld devices followed by selective chemical development with common reagents to generate micropatterns with depths/heights between 5-10 μm, which can be used to guide cell behavior. The remarkable straightforwardness of the process enables this class of microengineering techniques to be broadly accessible to diverse research communities. This article is protected by copyright. All rights reserved

Direct deep UV lithography to micropattern PMMA for stem cell culture

Microengineering is increasingly being used for controlling the microenvironment of stem cells. Here, a novel method for fabricating structures with subcellular dimensions in commonly available thermoplastic poly(methyl methacrylate) (PMMA) is shown. Microstructures are produced in PMMA substrates using Deep Ultraviolet lithography, and the effect of different developers is described. Microgrooves fabricated in PMMA are used for the neuronal differentiation of mouse embryonic stem cells (mESCs) directly on the polymer. The fabrication of 3D, curvilinear patterned surfaces is also highlighted. A 3D multilayered microfluidic chip is fabricated using this method, which includes a porous polycarbonate (PC) membrane as cell culture substrate. Besides directly manufacturing PMMA-based microfluidic devices, an application of the novel approach is shown where a reusable PMMA master is created for replicating microstructures with polydimethylsiloxane (PDMS). As an application example, microchannels fabricated in PDMS are used to selectively expose mESCs to soluble factors in a localized manner. The described microfabrication process offers a remarkably simple method to fabricate for example multifunctional topographical or microfluidic culture substrates outside cleanrooms, thereby using inexpensive and widely accessible equipment. The versatility of the underlying process could find various applications also in optical systems and surface modification of biomedical implants

Self-assembly of electrospun nanofibers into gradient honeycomb structures

The self-assembly approach is a technically simple, rapid, and direct way to realize selective deposition of electrospun nanofibers. In the present study, we aimed to fabricate gradient polycaprolactone (PCL) honeycomb meshes by electrospinning. We demonstrated for the first time the ability to effectively fabricate a self-assembled gradient honeycomb pattern in electrospun meshes. Different honeycomb patterns were successfully fabricated by controlling the electrospinning conditions. The working distance was found to be the most important factor for the formation of gradient honeycomb structures. At a smaller working distance of 12 cm, gradients honeycomb patterns were successfully fabricated. The pore diameter of the obtained gradient honeycomb structures spanned a range from 800 mu m to 300 mu m. The average depth of gradient honeycomb was 123 +/- 56 mu m. These findings are interesting and particularly useful for us to optimize the design of gradients honeycomb scaffolds for interface tissue regeneration. (c) 2019 The Authors. Published by Elsevier Ltd