Chemical transfection of cells in picoliter aqueous droplets in fluorocarbon oil.

Department of Chemistry, Nanjing University, Nanjing, PR China.
Analytical Chemistry (Impact Factor: 5.82). 10/2011; 83(22):8816-20. DOI: 10.1021/ac2022794
Source: PubMed

ABSTRACT The manipulation of cells inside water-in-oil droplets is essential for high-throughput screening of cell-based assays using droplet microfluidics. Cell transfection inside droplets is a critical step involved in functional genomics studies that examine in situ functions of genes using the droplet platform. Conventional water-in-hydrocarbon oil droplets are not compatible with chemical transfection due to its damage to cell viability and extraction of organic transfection reagents from the aqueous phase. In this work, we studied chemical transfection of cells encapsulated in picoliter droplets in fluorocarbon oil. The use of fluorocarbon oil permitted high cell viability and little loss of the transfection reagent into the oil phase. We varied the incubation time inside droplets, the DNA concentration, and the droplet size. After optimization, we were able to achieve similar transfection efficiency in droplets to that in the bulk solution. Interestingly, the transfection efficiency increased with smaller droplets, suggesting effects from either the microscale confinement or the surface-to-volume ratio.

1 Bookmark
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: An attractive option for tissue engineering is to use of multicellular spheroids as microtissues, particularly with stem cell spheroids. Conventional approaches of fabricating spheroids suffer from low throughput and polydispersity in size, and fail to supplement cues from extracellular matrix (ECM) for enhanced differentiation. In this study, we report the application of microfluidics-generated water-in-oil-in-water (w/o/w) double-emulsion (DE) droplets as pico-liter sized bioreactor for rapid cell assembly and well-controlled microenvironment for spheroid culture. Cells aggregated to form size-controllable (30-80 μm) spheroids in DE droplets within 150 min and could be retrieved via a droplet-releasing agent. Moreover, precursor hydrogel solution can be adopted as the inner phase to produce spheroid-encapsulated microgels after spheroid formation. As an example, the encapsulation of human mesenchymal stem cells (hMSC) spheroids in alginate and alginate-arginine-glycine-aspartic acid (-RGD) microgel was demonstrated, with enhanced osteogenic differentiation further exhibited in the latter case.
    Scientific Reports 01/2013; 3:3462. · 5.08 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A novel coaxial electrospray technology is developed to generate microcapsules with a hydrogel shell of alginate and an aqueous liquid core of living cells using two aqueous fluids in one step. Approximately 50 murine embryonic stem (ES) cells encapsulated in the core with high viability (92.3 ± 2.9%) can proliferate to form a single ES cell aggregate of 128.9 ± 17.4 μm in each microcapsule within 7 days. Quantitative analyses of gene and protein expression indicate that ES cells cultured in the miniaturized 3D liquid core of the core-shell microcapsules have significantly higher pluripotency on average than the cells cultured on the 2D substrate or in the conventional 3D alginate hydrogel microbeads without a core-shell architecture. The higher pluripotency is further suggested by their significantly higher capability of differentiation into beating cardiomyocytes and higher expression of cardiomyocyte specific gene markers on average after directed differentiation under the same conditions. Considering its wide availability, easiness to set up and operate, reusability, and high production rate, the novel coaxial electrospray technology together with the microcapsule system is of importance for mass production of ES cells with high pluripotency to facilitate translation of the emerging pluripotent stem cell-based regenerative medicine into the clinic.
    Integrative Biology 07/2014; · 4.32 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. Compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. As an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. Secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. The beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ∼200 Hz as well as cell enrichment. The microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ∼1 million cells, the microfluidic operations require 2-6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5-7 d.
    Nature Protocol 04/2013; 8(5):870-91. · 8.36 Impact Factor

Full-text (2 Sources)

Available from
Aug 14, 2014