Microfluidic-based patterning of embryonic stem cells for in vitro development studies

Abstract

In vitro recapitulation of mammalian embryogenesis and examination of the emerging behaviours of embryonic structures require both the means to engineer complexity and accurately assess phenotypes of multicellular aggregates. Current approaches to study multicellular populations in 3D configurations are limited by the inability to create complex (i.e. spatially heterogeneous) environments in a reproducible manner with high fidelity thus impeding the ability to engineer microenvironments and combinations of cells with similar complexity to that found during morphogenic processes such as development, remodelling and wound healing. Here, we develop a multicellular embryoid body (EB) fusion technique as a higher-throughput in vitro tool, compared to a manual assembly, to generate developmentally relevant embryonic patterns. We describe the physical principles of the EB fusion microfluidic device design; we demonstrate that >60 conjoined EBs can be generated overnight and emulate a development process analogous to mouse gastrulation during early embryogenesis. Using temporal delivery of bone morphogenic protein 4 (BMP4) to embryoid bodies, we recapitulate embryonic day 6.5 (E6.5) during mouse embryo development with induced mesoderm differentiation in murine embryonic stem cells leading to expression of Brachyury-T-green fluorescent protein (T-GFP), an indicator of primitive streak development and mesoderm differentiation during gastrulation. The proposed microfluidic approach could be used to manipulate hundreds or more of individual embryonic cell aggregates in a rapid fashion, thereby allowing controlled differentiation patterns in fused multicellular assemblies to generate complex yet spatially controlled microenvironments.

Controlling cell–cell interactions using surface acoustic waves

“…Despite significant advances in this field, to further understand how cells interact and communicate with each other, a robust, biocompatible method to precisely control the spatial and temporal association of cells and to create defined cellular assemblies is urgently needed (4). Although several methods have been used to pattern cells, limitations still exist for the demonstrated methods including those that make use of optical, electrical, magnetic, hydrodynamic, and contact printing technologies (5)(6)(7)(8)(9). Firstly, most of the methods require modification of the cell's native state.…”

In Vitro Microscale Models for Embryogenesis

“…[83] Suri et al designed a high-throughput in vitro multicellular EB fusion device to investigate the potential of introducing a primitive streak formation-like pattern within microenvironments of BMP4 (linked to mesoderm differentiation) (Figure 3C). [84] The authors built a reliable trapping microchip with high efficiency and reproducibility. Within cross-flow serpentine channels, two mouse EBs were trapped into fusion microarrays guided by fluid flow, and Brachyury-T-GFP expression (an early developmental marker associated with the primitive streak formation) in cells were monitored over time.…”

Advances in microfluidic platforms for analyzing and regulating human pluripotent stem cells

“…For example, Suri et al . developed a microfluidic trap to pattern and fuse two different types of EBs, one differentiated in the presence of BMP4 and the other in the absence of BMP4 [24]. The fused EBs exhibited spatial induction of mesoderm differentiation reminiscent of primitive streak formation, providing a system to study early embryonic morphogenesis in vitro .…”