A microfluidic design for desalination and selective removal and addition of components in biosamples.

We present the development of a microfluidic device that is able to selectively and nondisturbingly remove or add components to liquid samples, which allows control and conditioning of the samples for biomedical tests. The device consists of a series of chambers for sample retention and a through channel. Because smaller particles diffuse faster, small particles in the sample such as salt ions rapidly escape the chamber by diffusion and are subsequently removed by a carrier flow in the channel, leaving macromolecules of interest in the "desalted" solution. Conversely, components lacking in the sample can be diffused in by reversing the concentration gradient between the flow and the sample chamber. The ability to control the ionic strength of a sample offers many advantages in biological sample preparation as most biofluids contain high salt contents, making them unsuitable for downstream molecular analyses without additional sample treatments which could cause sample loss, contamination, and cost increase. Making use of the nature of laminar flow in a microfluidic device and mass transport by diffusion, we have developed an analytical model to calculate concentration profiles for different particles. Excellent agreements were found between the theory and the experiment, making the results highly reliable and predictable. Since the device and the principle is applicable to a wide range of biological samples, it can be incorporated into the workflow of various applications for research and in vitro diagnosis such as ion exchange, DNA sequencing, immuno assay, vesicle, cell secretion analysis, etc.

A single-cell translocation and secretion assay (TransSeA).

Understanding biological heterogeneity at the single cell level is required for advancing insights into the complexity of human physiology and diseases. While advances in technological and analytical methods have afforded unprecedented glimpses of this heterogeneity, the information captured to date largely represents one-time "snap" shots of single cell physiology. To address the limits of existing methods and to accelerate discoveries from single cell studies, we developed a single-cell translocation and secretion assay (TransSeA) that supports time lapse analysis, enables molecular cargo analysis of secretions such as extracellular vesicles (EVs) from single cells, allows massively parallel single cell transfer according to user-defined cell selection criteria, and supports tracking of phenotypes between parental and progeny cells derived from single cells. To demonstrate the unique capabilities and efficiencies of the assay, we present unprecedented single cell studies related to cell secretions, EV cargos and cell intrinsic properties. Although used as examples to demonstrate the feasibility and versatility of the technology, the studies already provided insights into key unanswered questions such as the microRNAs carried by EVs, the relationships between EV secretion rates and gene expressions, and the spontaneous, trans-generational phenotypic changes in EV secretion between parental and progeny cells.

A robust vitronectin-derived peptide for the scalable long-term expansion and neuronal differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (hNPCs).

Despite therapeutic advances, neurodegenerative diseases and disorders remain some of the leading causes of mortality and morbidity in the United States. Therefore, cell-based therapies to replace lost or damaged neurons and supporting cells of the central nervous system (CNS) are of great therapeutic interest. To that end, human pluripotent stem cell (hPSC) derived neural progenitor cells (hNPCs) and their neuronal derivatives could provide the cellular 'raw material' needed for regenerative medicine therapies for a variety of CNS disorders. In addition, hNPCs derived from patient-specific hPSCs could be used to elucidate the underlying mechanisms of neurodegenerative diseases and identify potential drug candidates. However, the scientific and clinical application of hNPCs requires the development of robust, defined, and scalable substrates for their long-term expansion and neuronal differentiation. In this study, we rationally designed a vitronectin-derived peptide (VDP) that served as an adhesive growth substrate for the long-term expansion of several hNPC lines. Moreover, VDP-coated surfaces allowed for the directed neuronal differentiation of hNPC at levels similar to cells differentiated on traditional extracellular matrix protein-based substrates. Overall, the ability of VDP to support the long-term expansion and directed neuronal differentiation of hNPCs will significantly advance the future translational application of these cells in treating injuries, disorders, and diseases of the CNS.

A chemically defined substrate for the expansion and neuronal differentiation of human pluripotent stem cell-derived neural progenitor cells.

Due to the limitation of current pharmacological therapeutic strategies, stem cell therapies have emerged as a viable option for treating many incurable neurological disorders. Specifically, human pluripotent stem cell (hPSC)-derived neural progenitor cells (hNPCs), a multipotent cell population that is capable of near indefinite expansion and subsequent differentiation into the various cell types that comprise the central nervous system (CNS), could provide an unlimited source of cells for such cell-based therapies. However the clinical application of these cells will require (i) defined, xeno-free conditions for their expansion and neuronal differentiation and (ii) scalable culture systems that enable their expansion and neuronal differentiation in numbers sufficient for regenerative medicine and drug screening purposes. Current extracellular matrix protein (ECMP)-based substrates for the culture of hNPCs are expensive, difficult to isolate, subject to batch-to-batch variations, and, therefore, unsuitable for clinical application of hNPCs. Using a high-throughput array-based screening approach, we identified a synthetic polymer, poly(4-vinyl phenol) (P4VP), that supported the long-term proliferation and self-renewal of hNPCs. The hNPCs cultured on P4VP maintained their characteristic morphology, expressed high levels of markers of multipotency, and retained their ability to differentiate into neurons. Such chemically defined substrates will eliminate critical roadblocks for the utilization of hNPCs for human neural regenerative repair, disease modeling, and drug discovery.