Enzyme-Free Dissociation of Neurospheres by a Microfluidic Chip-Based Method

Abstract

Neurosphere assay is a common and robust method for identification of neural stem/progenitor cells, but obtaining large numbers of live single cells from dissociated neurospheres is difficult using nonenzymatic methods. Here, we present an enzyme-free method for high-efficiency neurosphere dissociation into single cells using microfluidic device technology. This method allows single cell dissociation of DC115 and KT98 cells with high cell viabilities (80-85 %), single-cell yield (91-95 %), and recovery (75-93 %).

Microfluidic platforms for single neuron analysis

“…These chambers also maintain the high viability of dissociated single cells and retain their growth and differentiation features. The individual cells successively regrow neurospheres and shown to be differentiated into the three central neural lineages [ 22 ]. The optimization of microfluidic device design parameters (geometry and dimensions of the micro-pillars) and operational parameters (flow rate) improves the dissociation results [ 23 ].…”

“… Method Principle Modules Advantages Disadvantages Capture ratio/Single cell yield Cell viability Reported application for single neuron analysis Ref. Laser cell capture Laser capture micro-dissection Gravity-assisted Microdissection; Laser pressure catapult; Laser induced forward transfer Nullifies the effects of cytophilic/phobic surface Slow capture rate 1 cell/90s 85% Guidance axon regeneration [ [17] , [18] , [19] ] Microchannels Mechanical pressure Micropillars; Asymmetric channels; Layered microfluidics Avoids protease and collagenolytic activities, No immune response High flow rate causing cell damage 91–95% (80–85%) Axon growth [ 22 ] Microsieve device Capillary pumping, Hydrodynamic flow Passive pumping; Microsieve electrode array, Polymer replica moulded Label-free, Digital readout of signals at the single cell level, High parallelization, High spatial control Labor-intensive in absence of electrodes 80% 90% over 7 days Calcium imaging [ 14 , 26 , 30 ] Inertial microfluidics Inertial lift force (FL) and a curvature-induced Dean's drag force (FD) Differential inertial focusing; Selective inertial focusing Low shear stress, Low flow rates Inability to predict outcome, limited ability to deal with concentrated cellular samples 97% >90% Profiling neuro-chemistry occurring in selected neurons [ 24 ] Di-electophoresis Di-electrophoretic force Non-transparent electrodes; transparent indium-tin-oxide (ITO) based Non-invasive, Label-free and non-destructive, Improved homeostatic conditions reduced, contamination risk Confound live-cell imaging 100 99% at 5 days Electrophysiological recording and neurological studies. [ ...…”