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

Abstract

Embryoid Bodies (EBs) are large (> 100 μm) 3D microtissues composed of stem cells, differentiating cells and extracellular matrix (ECM) proteins that roughly recapitulate early embryonic development. EBs are widely used as in vitro model systems to study stem cell differentiation and the complex physical and chemical interactions contributing to tissue development. Though much has been learned about differentiation from EBs, the practical and technical difficulties of effectively probing and properly analyzing these 3D microtissues has limited their utility and further application. We describe advancement of a technology platform developed in our laboratory, multiphoton flow cytometry (MPFC), to detect and sort large numbers of intact EBs based on size and fluorescent reporters. Real-time and simultaneous measurement of size and fluorescence intensity are now possible, through the implementation of image processing algorithms in the MPFC software. We applied this platform to purify populations of EBs generated from murine induced pluripotent stem (miPS) cells exhibiting enhanced potential for cardiomyocyte differentiation either as a consequence of size or expression of NKX2-5, a homeodomain protein indicative of precardiac cells. Large EBs (330-400 μm, diameter) purified soon after EB formation showed significantly higher potential to form cardiomyocytes at later time points than medium or small EBs. In addition, EBs expressing NKX2-5 soon after EB formation were more likely to form beating areas, indicative of cardiomyocyte differentiation, at later time points. Collectively, these studies highlight the ability of the MPFC to purify EBs and similar microtissues based on preferred features exhibited at the time of sorting or on features indicative of future characteristics or functional capacity.

Noninvasive sorting of stem cell aggregates based on intrinsic markers

“…Treated and untreated populations were analyzed separately on the MPFC before the sorting trials to determine each respective distribution of NADH fluorescence intensity. A real‐time fluorescence intensity calculation (as previously described) was used to determine total NADH expression per EB. Briefly, bright field images of EBs were used to identify the borders of EBs and total NADH fluorescence confined by this border was determined, thereby accounting for the non‐contiguous distribution of intensity in each EB.…”

On Chip Sorting of Stem Cell-Derived β Cell Clusters Using Traveling Surface Acoustic Waves

“…Microfluidic devices are usually more cost-effective to fabricate, easier to transport, and capable of integrating complex operations (such as micromixing) more effectively than their large-scale counterparts. Though extensive research efforts toward cell size-based sorting have been made, efforts for sorting islets and other cell clusters by size have been relatively more limited. − …”

“…Redesigning and refabricating a new device with a different cutoff size according to the clinical need can be laborious and expensive. To address this concern, Buschke et al devised an active image detection-based microfluidic device for sorting EBs (100–400 μm) on the basis of size and fluorescence intensity. , The sorting throughput of this device was limited due to the low scan speed (∼5 frames/s), long actuation time (0.1–1 s), and necessary time delay (1–3 s) in the system due to use of an actuation mechanism based on solenoid valves.…”