Single Gel Bead Research Articles

Label-free single-cell analysis in microdroplets using a light-scattering-based optofluidic chip

Droplet-based single-cell analysis is a very powerful tool for studying phenotypic and genomic heterogeneity at single-cell resolution for a variety of biological problems. In conventional two-phase droplet microfluidics, due to the mismatch in optical properties between oil and aqueous phases, light scattering mainly happens at the oil/water interface that disables light-scattering-based cell analysis confined in microdroplets. Detection and analysis of cells in microdroplets thus mostly rely on the fluorescence labeling of cell samples, which may suffer from complex operation, cytotoxicity, and low fluorescence stability. In this work, we propose a novel light-scattering-based droplet screening (LSDS) that can effectively detect and characterize single cells confined in droplets by adjusting the optical properties of droplets in a multiangle optofluidic chip. Theoretical and simulated calculations suggest that refractive index (RI) matching in droplet two-phase materials can reduce or eliminate droplets’ scattered signals (background signal), enabling the differentiation of scattered signals from single cells and particles within droplets. Furthermore, by using a set of multiangle (from −145° to 140°) optical fibers integrated into the optofluidic chip, the scattered light properties of droplets with the RI ranging from 1.334 to 1.429 are measured. We find that the smaller the RI and size of microparticles inside droplets are, the smaller the RI difference between two-phase materials Δn is required. Especially, when Δn is smaller than 0.02, single cells in droplets can be detected and analyzed solely based on light scattering. This capability allows to accurately detect droplets containing one single cell and one single gel bead, a typical droplet encapsulation for single-cell sequencing. Altogether, this work provides a powerful platform for high-throughput label-free single-cell analysis in microdroplets for diverse single-cell related biological assays.

Microbial growth rates and local external mass transfer coefficients in a porous bed biofilm system measured by 19 F magnetic resonance imaging of structure, oxygen concentration, and flow velocity.

19 F nuclear magnetic resonance (NMR) oximetry and 1 H NMR velocimetry were used to noninvasively map oxygen concentrations and hydrodynamics in space and time in a model packed bed biofilm system in the presence and absence of flow. The development of a local oxygen sink associated with a single gel bead inoculated with respiring Escherichia coli was analyzed with a phenomenological model to determine the specific growth rate of the bacteria in situ, returning a value (0.66 hr-1 ) that was close to that measured independently in planktonic culture (0.62 hr-1 ). The decay of oxygen concentration in and around the microbiologically active bead was delayed and slower in experiments conducted under continuous flow in comparison to no-flow experiments. Concentration boundary layer thicknesses were determined and Sherwood numbers calculated to quantify external mass transfer resistance. Boundary layers were thicker in no-flow experiments compared to experiments with flow. Whereas the oxygen concentration profile across a reactive biofilm particle was symmetric in no-flow experiments, it was asymmetric with respect to flow direction in flow experiments with Sherwood numbers on the leading edge (Sh = 7) being larger than the trailing edge (Sh = 3.5). The magnitude of the experimental Sh was comparable to values predicted by a variety of correlations. These spatially resolved measurements of oxygen distribution in a geometrically complex model reveal in innovative detail the local coupling between microbial growth, oxygen consumption, and external mass transfer.

Abstract 150: Single cell mRNA quantification from 1000s of cells in healthy and malignant tumor samples using a high-throughput droplet-based system

Abstract Advances in single cell RNA quantification techniques have enabled comprehensive study of subpopulations of cells within a heterogeneous population. The application of single cell quantification techniques to oncology is helping to elucidate the complex variability in genetic and epigenetic interactions that occur within tumor cells and their microenvironment. However, current single cell RNA-sequencing methods are limited by their reliance on costly infrastructure and laborious experimental protocols. We developed the GemCode Platform, which combines microfluidics with molecular barcoding and custom bioinformatics software to enable 3’ mRNA counting from thousands of single cells. Here we utilized the GemCode Platform to profile primary cells from healthy donors and cancer patients. Cell lines and cancer samples were obtained from commercial sources. Single cells, reagents and a single gel bead containing barcoded oligonucleotides were encapsulated into picoliter-sized droplets using the 10X Genomics GemCode Platform. The platform achieved extremely high cell loading efficiency (> 50%), enabling the creation of libraries from precious samples. Lysis and barcoded reverse transcription of RNAs from single cells were performed inside each droplet. High quality next generation sequencing libraries were finished in a single bulk reaction. The GemCode software suite was utilized for processing, interactive analysis and visualization of single cell gene expression data. We demonstrated single cell behavior through mouse- and human cell mixing experiments with a low doublet rate of <1%, making the platform suitable for profiling of rare cancer cell populations. We profiled >40,000 peripheral blood mononuclear cells from healthy donors and detected all major subpopulations (i.e., B cells, CD4+ T cells, CD8+ T cells, NK cells, dendritic cells, monocytes) in similar proportions to those previously reported in the literature. Notably, the high-throughput nature of the platform enabled resolution of finer sub-structures such as CD4+ effector memory cells and CD4+ central memory cells. Experiments comparing cells isolated from patients with hematologic malignancies (such as CLL, AML and CML) with whole bone marrow from healthy donors further demonstrate the power of single cell profiling for characterizing disease-associated changes in complex tissues. We demonstrate the ability to perform high-throughput gene expression profiling of mRNAs in single cells. The high-throughput platform enables detection of rare cells in a heterogeneous tumor population. Moreover, efficient cell loading enables analysis of clinically relevant sample types with limited cell input. An integrated single cell mRNA analysis will lead to novel insights into the molecular characteristics of individual cancer cells and provide targets for therapeutic intervention. Citation Format: Grace X.y. Zheng, Tarjei Mikkelsen, Jessica Terry, Phillip Belgrader, Paul Ryvkin, Ryan Wilson, Tobias D. Wheeler, Zachary Bent, Geoff McDermott, Solongo Ziraldo, Alexander Wong, Michael Schnall-Levin, Ben Hindson. Single cell mRNA quantification from 1000s of cells in healthy and malignant tumor samples using a high-throughput droplet-based system. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 150.

Ion-Exchange Controls the Kinetics of Deswelling of Polyelectrolyte Microgels in Solutions of Oppositely Charged Surfactant

The kinetics of deswelling of sodium polyacrylate microgels (radius 30-140 microm) in aqueous solutions of dodecyltrimethylammonium bromide is investigated by means of micropipet-assisted light microscopy. The purpose of the study is to test a recent model (J. Phys. Chem. B 2003, 107, 9203) proposing that the rate of the volume change is controlled by the transport of surfactant from the solution to the gel core (ion exchange) via the surfactant-rich surface phase appearing in the gel during the volume transition. Equilibrium swelling characteristics of the gel network in surfactant-free solutions and with various amounts of surfactant present are presented and discussed with reference to related systems. A relationship between gel volume and degree of surfactant binding is determined and used in theoretical predictions of the deswelling kinetics. Experimental data for single gel beads observed during deswelling under conditions of forced convection are presented and compared with model calculations. It is demonstrated that the dependences of the kinetics on initial gel size, the surfactant concentration in the solution, and the liquid flow rate are well accounted for by the model. It is concluded that the deswelling rates of the studied gels are strongly influenced by the mass transport of surfactant between gel and solution (stagnant layer diffusion), but only to a minor extent by the transport through the surface phase. The results indicate that, during the volume transition, swelling equilibrium (network relaxation/transport of water) is established on a relatively short time scale and, therefore, can be treated as independent of the ion-exchange kinetics. Theoretical aspects of the kinetics and mechanisms of surfactant transport through the surface phase are discussed.

Fluidized-bed and packed-bed characteristics of gel beads

Abstract A liquid-fluidized bed or packed bed with gel beads is attractive as an immobilized-cell bioreactor. The performance of such bioreactors is influenced by the physical behavior of these beads. Three different but related aspects involving the drag force between particles and liquid were studied for five types of gel beads, differing in diameter and density: (1) the terminal settling velocity of a single gel bead, (2) the pressure drop over a packed bed and (3) the voidage in a liquid-fluidized bed. Qualitatively, the same trends in these aspects were observed for gel beads as for conventional solids. Quantitatively, however, these aspects were incorrectly predicted by established models (with one exception). It was found that the drag force between gel bead and flowing liquid is smaller than that for conventional solids. As an explanation, two hypotheses are suggested. The first one attributes the drag reduction to small amounts of dissolved polymer. The second one attributes the smaller drag force to the surface nature of gel beads: gel beads contain over 95% of water and thus can be regarded as ‘rigid’ water droplets. Hence, the gel bead surface might show water-like properties. As an alternative to drag-coefficient relations for conventional solids, the drag coefficient of a single gel bead in a packed or fluidized bed could successfully be described by adapting an existing relation. The success of this description facilitates a more rational design of packed and fluidized beds of gel beads.

Diffusion of globular proteins in a Sephadex G200 gel bead

We measured FRAP curves of solutions of globular proteins fractions labeled with fluorescein and included in a Sephadex G 200 single gel bead. We determined the diffusion coefficient D of these macromolecules in the gel. The measurement of the partition coefficient of these labeled macromolecules showed that fluorescein residues can induce a partial adsorption of the macromolecules on the gel matrix. When corrected for this effect, the reduced coefficient of diffusion D/D 0 , (where D 0 is the diffusion coefficient in the free solution), was found higher than the value predicted by the Ogston et al. theory (Proc. Roy. Soc. A333 (1973) 297). This discrepancy was ascribed to the dynamic flexibility of the Sephadex gel matrix.

Scanning Microfluorimetry of Ca-Alginate Immobilized Zymomonas Mobilis

Scanning microfluorimetric techniques offer a means for in situ investigation of spatial heterogeneities in biomass density and cell composition in macroscopic, aggregate-cell systems such as immobilized-cell biocatalysts, biofilms, mold pellets and tumors. Much of the analytical power of flow cytometry, with the important addition of spatial resolution, can be brought to bear on the study of these systems. The utility of these techniques is demonstrated for the experimental determination of spatial gradients in biomass density and bio-catalytic activity in Ca-alginate beads containing an entrapped, living bacterium, Zymomonas mobilis ATCC 10988. Using cell RNA content as an indicator of cell growth rate, scanning microfluorimetry data indicates that immobilized cells in a single gel bead can exhibit the fullest possible variation in specific growth rates, from maximal to resting. Thus, clear gradients in biological growth rate within such biocatalysts are quantifiable, with implications for any fully or partially growth associated products.

Analysis of dispersion mechanism in gel chromatography. II. Dispersion mechanism in gel chromatography of proteins.

The dispersion mechanism in gel chromatography of proteins was quantitatively studied on the basis of a dispersion model which gave consideration to the contribution of gel phase diffusion and longitudinal dispersion in a column. Particularly, the effect of gel phase diffusion was examined in detail by comparing the values determined by applying the moment analysis to the elution curve and those obtained for a single gel bead.When the distribution coefficient of solute was small and the particle size of gel was large, the elution curve was asymmetrical and had a prolonged tail of low concentration. The gel phase diffusion coefficient determined from the elution curve was much higher than that for a single gel bead owing to the effect of the tail. This suggested that only a small portion near the gel bead surface was accessible to most of the solute applied owing to the much lower diffusion velocity in gel than the superficial velocity. The column efficiency improved as compared with the theoretical result...