High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays

Abstract

Heterogeneity in cell populations poses a major obstacle to understanding complex biological processes. Here we present a microfluidic platform containing thousands of nanoliter-scale chambers suitable for live-cell imaging studies of clonal cultures of nonadherent cells with precise control of the conditions, capabilities for in situ immunostaining and recovery of viable cells. We show that this platform mimics conventional cultures in reproducing the responses of various types of primitive mouse hematopoietic cells with retention of their functional properties, as demonstrated by subsequent in vitro and in vivo (transplantation) assays of recovered cells. The automated medium exchange of this system made it possible to define when Steel factor stimulation is first required by adult hematopoietic stem cells in vitro as the point of exit from quiescence. This technology will offer many new avenues to interrogate otherwise inaccessible mechanisms governing mammalian cell growth and fate decisions.

Stationary nanoliter droplet array with a substrate of choice for single adherent/nonadherent cell incubation and analysis

“…There are methods that bypass the production and capturing under flow altogether by initially producing stationary plugs within the geometry of the device (2,(23)(24)(25). However, upon their adjustment to prolonged mammalian assays, they require multilayer fabrication, either involving high-pressure cell loading or not allowing for different substrates to be used (26,27). Several single-cell platforms that encapsulate cells in stationary droplet arrays are available (5,(28)(29)(30)(31)(32)(33)(34)(35); however, the ability to culture adherent cells for long periods remains difficult (36).…”

Technology Advancement for Integrative Stem Cell Analyses

“…The maturation of microfluidics from its infancy into adolescence over the past 10 years has yielded many approaches to trap cells and manipulate their microenvironments in defined ways. [55][56][57][58][59] The simplest strategy for isolating cells involves the placement of weirs or traps that lie in line with the fluid flow. [60][61][62] The pressure exerted by the flowing fluids holds the cells in place for imaging cytometry and the measurement of some dynamic properties, including calcium flux on stimulation, 63 cell motility, 64 or proliferation.…”

“…The advantage of this approach is that a cell flowing in the fluidic channel is trapped. To obtain sophisticated controlling of the 58 Hong et al, 59 Brouzes et al, 83 and Moeller et al 90 Color images available online at www.liebertpub.com/teb cell arrays, hydrodynamic effects are applied with an external field gradient such as optical, electromagnetic, and acoustic for feasible confinement of cells. [76][77][78] However, the narrow dimensions of the channels near the valves (usually <10 mm) have limited their use in these systems for the extended analyses of stem cells.…”

Recent developments in microfluidic large scale integration

“…As fluid volumes decrease, evaporation becomes a challenge, requiring hydration elements [31]. When chip complexity increases, on-chip nozzles [32 ] can assist with product recovery.…”