Flip channel: A microfluidic device for uniform-sized embryoid body formation and differentiation

Abstract

This paper reports a two-layered polydimethylsiloxane microfluidic device-Flip channel, capable of forming uniform-sized embryoid bodies (EBs) and performing stem cell differentiation within the same device after flipping the microfluidic channel. The size of EBs can be well controlled by designing the device geometries, and EBs with multiple sizes can be formed within a single device to study EB size-dependent stem cell differentiation. During operation of the device, cells are positioned in the designed positions. As a result, observation and monitoring specific population of cells can be achieved for further analysis. In addition, after flipping the microfluidic channel, stem cell differentiation from the EBs can be performed on an unconfined flat surface that is desired for various differentiation processes. In the experiments, murine embryonic stem cells (ES-D3) are cultured and formed EBs inside the developed device. The size of EBs is well controlled inside the device, and the neural differentiation is performed on the formed EBs after flipping the channel. The EB size-dependent stem cell differentiation is studied using the device to demonstrate its functions. The device provides a useful tool to study stem cell differentiation without complicated device fabrication and tedious cell handling under better-controlled microenvironments.

Novel RNA Viral Vectors for Chemically Regulated Gene Expression in Embryonic Stem Cells

“…A notable observation was that 2mu-VSV appeared to be silenced in the peripheral cells of EBs in the culture where the outgrowth of differentiated cells is expected. 25,26 We speculate that the maturation of the type I interferon pathway or other antiviral pathways in the differentiated cells clears VSV, as the differentiation progresses. This is a favorable property of 2mu-VSV for future applications as a transgene vector to induce stem cell differentiation.…”

Conceptual and Experimental Tools to Understand Spatial Effects and Transport Phenomena in Nonlinear Biochemical Networks Illustrated with Patchy Switching

“…In another example, at larger scales, the direction of stem cell differentiation was shown to be potentiated by the size of stem cell embryoid bodies generated in microfluidic wells (3,27). Studies by two independent groups have shown that small embryoid bodies remained inert to neurodifferentiation, whereas larger clusters (>250-350 μm in diameter) adopted a neural morphology with extensive neurite formation (Figure 3c) (27,31). The mechanism of this is still unclear and may or may not be due to diffusible signals, although the role of diffusible stem cell signals is being actively investigated (22,32).…”

“…Finally, true 3D patterning is now possible as a result of advances in biomaterials, chemistry, and 3D bioprinting, so that molecules and cells can be patterned on or within functionalized polymeric scaffolds or matrices for more realistic, tissue-like geometries (41)(42)(43)(44)(45). These patterning tools have been invaluable for evaluating the effect of patch size on blood coagulation (Figure 3a) (25) and stem cell differentiation (Figure 3c) (27,31,(46)(47)(48). Patterning can also distinguish the role of cell-cell contacts from that of soluble signaling (28) to help assess the extent to which transport-mediated signals drive a particular system.…”

A microfluidic trap array for longitudinal monitoring and multi-modal phenotypic analysis of individual stem cell aggregates

“…Next, devices were rinsed with sterile phosphate-buffered saline (PBS) and then incubated with a 1 mg/ml bovine serum albumin (BSA; Millipore) solution for 30 minutes to passivate the surfaces. 33, 48 A 200 μL pipette tip filled with cell culture medium was placed at the device inlet for aggregate loading and perfusion. Aggregates were collected and resuspended at a concentration of approximately 600 aggregates/mL in a solution of 1.05 g/mL Percoll (Sigma Aldrich) in phosphate-buffered saline (PBS) to prevent rapid settling.…”