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.

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. An economical and easily accessible method is developed and validated 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 and 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.

Thin fluorinated polymer film microcavity arrays for 3D cell culture and label-free automated feature extraction.

There is an increasing need for automated label-free morphometric analysis using brightfield microscopy images of 3D cell culture systems. This requires automated feature detection which can be achieved by improving the image contrast, e.g. by reducing the refractive index mismatch in the light path. Here, a novel microcavity platform fabricated using microthermoforming of thin fluorinated ethylene-propylene (FEP) films which match the refractive index of cell culture medium and provide a homogenous background signal intensity is described. FEP is chemically inert, mechanically stable and has been used as a substrate for light sheet microscopy. The microcavities promote formation of mouse embryonic stem cell (mESC) aggregates, which show axial elongation and germ layer specification similar to embryonic development. A label-free feature extraction pipeline based on a machine-learning plugin for FIJI is used to extract morphometric features from time-lapse imaging in a highly robust and reproducible manner. Lastly, the pipeline is utilized for testing the effect of the drug Latrunculin A on the mESC aggregates, highlighting the platform's potential for high-content screening (HCS) in drug discovery. This new microengineered tool is an important step towards label-free imaging of free-floating stem cell aggregates and paves the way for high-content drug testing and translational studies.

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.

Fabrication of a self-assembled honeycomb nanofibrous scaffold to guide endothelial morphogenesis.

Controlling angiogenesis within tissue engineered constructs remains a critical challenge, especially with regard to the guidance of pre-vascular network formation. Here, we aimed to regulate angiogenesis on a self-assembled honeycomb nanofibrous scaffold. Scaffolds with honeycombs patterns have several desirable properties for tissue engineering, including large surface area, high structural stability and good permeability. Furthermore, the honeycomb pattern resembles early vascular network formation. The self-assembly electrospinning approach to honeycomb scaffolds is a technically simple, rapid, and direct way to realize selective deposition of nanofibers. To evaluate cell compatibility, spreading, proliferation and tube formation, human umbilical vein endothelial cells (HUVECs) were cultured on honeycomb scaffolds, as well as on random scaffolds for comparison. The optimized honeycomb nanofibrous scaffolds were observed to better support cell proliferation and network formation, which can facilitate angiogenesis. Moreover, HUVECs cultured on the honeycomb scaffolds were observed to reorganize their cell bodies into tube-like structures containing a central lumen, while this was not observed on random scaffolds. This work has shown that the angiogenic response can be guided by honeycomb scaffolds, allowing improved early HUVECs organization. The guided organization via honeycomb scaffolds can be utilized for tissue engineering applications that require the formation of microvascular networks.

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.