High-throughput physical phenotyping of cell differentiation

In this report,we present multiparameter deformability cytometry(m-DC),in which we explore a large set of parameters describing the physical phenotypes of pluripotent cells and their derivatives.m-DC utilizes microfluidic inertial focusing and hydrodynamic stretching of single cells in conjunction with high-speed video recording to realize high-throughput characterization of over 20 different cell motion and morphology-derived parameters.Parameters extracted from videos include size,deformability,deformation kinetics,and morphology.We train support vector machines that provide evidence that these additional physical measurements improve classification of induced pluripotent stem cells,mesenchymal stem cells,neural stem cells,and their derivatives compared to size and deformability alone.In addition,we utilize visual interactive stochastic neighbor embedding to visually map the high-dimensional physical phenotypic spaces occupied by these stem cells and their progeny and the pathways traversed during differentiation.This report demonstrates the potential of m-DC for improving understanding of physical differences that arise as cells differentiate and identifying cell subpopulations in a label-free manner.Ultimately,such approaches could broaden our understanding of subtle changes in cell phenotypes and their roles in human biology.

Particle focusing by 3D inertial microfluidics

Three-dimensional(3D)particle focusing in microfluidics is a fundamental capability with a wide range of applications,such as on-chip flow cytometry,where high-throughput analysis at the single-cell level is performed.Currently,3D focusing is achieved mainly in devices with complex layouts,additional sheath fluids,and complex pumping systems.In this work,we present a compact microfluidic device capable of 3D particle focusing at high flow rates and with a small footprint,without the requirement of external fields or lateral sheath flows,but using only a single-inlet,single-outlet microfluidic sequence of straight channels and tightly curving vertical loops.This device exploits inertial fluidic effects that occur in a laminar regime at sufficiently high flow rates,manipulating the particle positions by the combination of inertial lift forces and Dean drag forces.The device is fabricated by femtosecond laser irradiation followed by chemical etching,which is a simple two-step process enabling the creation of 3D microfluidic networks in fused silica glass substrates.The use of tightly curving three-dimensional microfluidic loops produces strong Dean drag forces along the whole loop but also induces an asymmetric Dean flow decay in the subsequent straight channel,thus producing rapid cross-sectional mixing flows that assist with 3D particle focusing.The use of out-of-plane loops favors a compact parallelization of multiple focusing channels,allowing one to process large amounts of samples.In addition,the low fluidic resistance of the channel network is compatible with vacuum driven flows.The resulting device is quite interesting for high-throughput on-chip flow cytometry.

Numerical investigation of the dynamics of a rigidspherical particle in a vortical cross-slot flow atmoderate inertia

The study of flow and particle dynamics in microfluidic cross-slot channels is of high relevance for lab-on-a-chipapplications. In this work, we investigate the dynamics of a rigid spherical particle in a cross-slot junction for a channelheight-to-width ratio of 0.6 and at a Reynolds number of 120 for which a steady vortex exists in the junction area.Using an in-house immersed-boundary-lattice-Boltzmann code, we analyse the effect of the entry position of theparticle in the junction and the particle size on the dynamics and trajectory shape of the particle. We find that thedynamics of the particle depend strongly on its lateral entry position in the junction and weakly on its vertical entryposition;particles that enter close to the centre show trajectory oscillations. Larger particles have longer residencetimes in the junction and tend to oscillate less due to their confinement. Our work contributes to the understanding ofparticle dynamics in intersecting flows and enables the design of optimised geometries for cytometry and particlemanipulation.

Computational cytometer based on magnetically modulated coherent imaging and deep learning

Detecting rare cells within blood has numerous applications in disease diagnostics.Existing rare cell detection techniques are typically hindered by their high cost and low throughput.Here,we present a computational cytometer based on magnetically modulated lensless speckle imaging,which introduces oscillatory motion to the magneticbead-conjugated rare cells of interest through a periodic magnetic force and uses lensless time-resolved holographic speckle imaging to rapidly detect the target cells in three dimensions(3D).In addition to using cell-specific antibodies to magnetically label target cells,detection specificity is further enhanced through a deep-learning-based classifier that is based on a densely connected pseudo-3D convolutional neural network(P3D CNN),which automatically detects rare cells of interest based on their spatio-temporal features under a controlled magnetic force.To demonstrate the performance of this technique,we built a high-throughput,compact and cost-effective prototype for detecting MCF7 cancer cells spiked in whole blood samples.Through serial dilution experiments,we quantified the limit of detection(LoD)as 10 cells per millilitre of whole blood,which could be further improved through multiplexing parallel imaging channels within the same instrument.This compact,cost-effective and high-throughput computational cytometer can potentially be used for rare cell detection and quantification in bodily fluids for a variety of biomedical applications.

Shaped 3D microcarriers for adherent cell culture and analysis

Standard tissue culture of adherent cells is known to poorly replicate physiology and often entails suspending cells in solution for analysis and sorting,which modulates protein expression and eliminates intercellular connections.To allow adherent culture and processing in flow,we present 3D-shaped hydrogel cell microcarriers,which are designed with a recessed nook in a first dimension to provide a tunable shear-stress shelter for cell growth,and a dumbbell shape in an orthogonal direction to allow for self-alignment in a confined flow,important for processing in flow and imaging flow cytometry.We designed a method to rapidly design,using the genetic algorithm,and manufacture the microcarriers at scale using a transient liquid molding optofluidic approach.The ability to precisely engineer the microcarriers solves fundamental challenges with shear-stress-induced cell damage during liquid-handling,and is poised to enable adherent cell culture,in-flow analysis,and sorting in a single format.

Single-cell sorting based on secreted products for functionally defined cell therapies

Cell therapies have emerged as a promising new class of“living”therapeutics over the last decade and have been particularly successful for treating hematological malignancies.Increasingly,cellular therapeutics are being developed with the aim of treating almost any disease,from solid tumors and autoimmune disorders to fibrosis,neurodegenerative disorders and even aging itself.However,their therapeutic potential has remained limited due to the fundamental differences in how molecular and cellular therapies function.While the structure of a molecular therapeutic is directly linked to biological function,cells with the same genetic blueprint can have vastly different functional properties(e.g.,secretion,proliferation,cell killing,migration).Although there exists a vast array of analytical and preparative separation approaches for molecules,the functional differences among cells are exacerbated by a lack of functional potency-based sorting approaches.In this context,we describe the need for next-generation single-cell profiling microtechnologies that allow the direct evaluation and sorting of single cells based on functional properties,with a focus on secreted molecules,which are critical for the in vivo efficacy of current cell therapies.We first define three critical processes for single-cell secretion-based profiling technology:(1)partitioning individual cells into uniform compartments;(2)accumulating secretions and labeling via reporter molecules;and(3)measuring the signal associated with the reporter and,if sorting,triggering a sorting event based on these reporter signals.We summarize recent academic and commercial technologies for functional single-cell analysis in addition to sorting and industrial applications of these technologies.These approaches fall into three categories:microchamber,microfluidic droplet,and lab-on-a-particle technologies.Finally,we outline a number of unmet needs in terms of the discovery,design and manufacturing of cellular therapeutics and how the next generation of single-cell functional screening technologies could allow the realization of robust cellular therapeutics for all patients.