Microfluidic sorting of microtissues

Abstract

Increasingly, in vitro culture of adherent cell types utilizes three-dimensional (3D) scaffolds or aggregate culture strategies to mimic tissue-like, microenvironmental conditions. In parallel, new flow cytometry-based technologies are emerging to accurately analyze the composition and function of these microtissues (i.e., large particles) in a non-invasive and high-throughput way. Lacking, however, is an accessible platform that can be used to effectively sort or purify large particles based on analysis parameters. Here we describe a microfluidic-based, electromechanical approach to sort large particles. Specifically, sheath-less asymmetric curving channels were employed to separate and hydrodynamically focus particles to be analyzed and subsequently sorted. This design was developed and characterized based on wall shear stress, tortuosity of the flow path, vorticity of the fluid in the channel, sorting efficiency and enrichment ratio. The large particle sorting device was capable of purifying fluorescently labelled embryoid bodies (EBs) from unlabelled EBs with an efficiency of 87.3% 6 13.5%, and enrichment ratio of 12.2 6 8.4 (n ¼ 8), while preserving cell viability, differentiation potential, and long-term function. V C 2012 American Institute of Physics. [http://dx

Large particle multiphoton flow cytometry to purify intact embryoid bodies exhibiting enhanced potential for cardiomyocyte differentiation

“…Sorting efficiency was defined as the ratio of the number of EBs physically collected from the sorting port, to the number of positive events detected in flow. A sorting efficiency of 89.0 ± 11.1% and an enrichment ratio of 25.4 ± 19.3 was achieved (Figure 3D), which are comparable to previously reported values corresponding to the purification of large fluorescent beads or EBs uniformly stained with CellTracker™ (Molecular Probes/Invitrogen, Carlsbad CA) dye 17 .…”

“…Previously, we used the MPFC to sort EBs based on contiguous fluorescence intensity within an EB 17 so that EBs could be effectively distinguished from fluorescent noise. This was accomplished by coupling our in house developed laser scanning acquisition software, WiscScan, with ImageJ-based 18 analysis tools for real time large particle analysis in flow.…”

“…The MPFC technology described here has advantages for sorting EBs based on size or reporter elements over previously described systems 24, 26 , including our first generation MPFC device 13, 17 . Our first generation device was accessible to laboratories with access to microfabrication capabilities and imaging systems with real time data acquisition capabilities.…”

Advances in microfluidic cell separation and manipulation

“…To obtain blur-free images with preserved spatial resolution at high speeds, ultra-fast imaging technology has been coupled with real-time image processing [10••]. Other recent advances in µFACS include the development of systems for sorting larger and irregularly shaped objects such as cell aggregates [11], embryos [12], and organisms such as the nematode Caenorhabditis elegans [13••,14]. The major contribution of these approaches is that information is spatially specific (e.g.…”

Endogenous Optical Signals Reveal Changes of Elastin and Collagen Organization During Differentiation of Mouse Embryonic Stem Cells

“…[31][32][33] Leukemia inhibitory factor (Millipore) and bone morphogenetic protein 4 (R&D Systems) were added to media at 2000 U/ mL and 10 ng/mL, respectively, to maintain pluripotency. 34 Mouse EBs were prepared using the hanging drop method.…”