An aqueous two phase system (ATPS) is composed of binary hydrophilic polymers, for example, polyethylene glycol (PEG) and dextran, under an immiscible condition, and can also exhibit micro-segregation to produce cell-sized microcompartments like water-in-water microdroplets. Without membranes, interestingly, the microdroplet can serve as a micro-vessel (reactor) that contains various biochemical macromolecules like DNAs and proteins. We here present that PEG/dextran ATPS micro-segregation can provide an effective soft boundary to separate these biochemical macromolecules from the external environment. Trapped DNAs and proteins were concentrated inside such small spaces, and therefore, their interaction could be highly promoted to cause passive aggregation and controlled cross-linking if a certain cross-linker was added. We believe that the ATPS microdroplets might be associated with complicated structures and functions of living cells.

Due to their aqueous environment and biocompatible polymers, aqueous two-phase systems (ATPS) provide a mild environment for partition of cells. A comprehensive understanding of cell partition in ATPS will facilitate cell separation and fractionation in ATPS for downstream cell-based analytical applications and various cellular and molecular biology studies. We report the effect of molecular weight of phase polymers on partition of cells between two aqueous phases and their interface. We generate three different ATPS by dissolving polyethylene glycol and dextran of different molecular weights in a standard cell culture medium of fixed composition. After suspending cells in each ATPS, we quantify the number of cells partitioned into each segregated phase and the interface between the two phases. Importantly, we use two-phase solutions of an identical interfacial tension from each ATPS to avoid the effect of interfacial tension on cell partition. Our results indicate that decreasing the molecular weight of one of the phase polymers results in distribution of a greater number of cells toward the phase rich in that polymer. Regardless of molecular weight of polymers used, two-phase solutions made with higher concentrations of polymers cause a significant shift toward cell partition to the interface. This study elucidates the role of polymer molecular weight on cell partition in ATPS and offers formulations for rapid and effective cell partition.

Aqueous two-phase systems and related emulsion-based structures defined within micro- and nanoscale environments enable a bottom-up synthetic biological approach to mimicking the dynamic compartmentation of biomaterial that naturally occurs within cells. Model systems we have developed to aid in understanding these phenomena include on-demand generation and triggering of reversible phase transitions in ATPS confined in microscale droplets, morpho-logical changes in networks of femtoliter-volume aqueous droplet interface bilayers (DIBs) formulated in microfluidic channels, and temperature-driven phase transitions in interfacial lipid bilayer systems supported on micro and nanostructured substrates. For each of these cases, the dynamics were intimately linked to changes in the chemical potential of water, which becomes increasingly susceptible to confinement and crowding. At these length scales, where interfacial and surface areas predominate over compartment volumes, both evaporation and osmotic forces become enhanced relative to ideal dilute solutions. Consequences of confinement and crowding in cell-sized microcompartments for increasingly complex scenarios will be discussed, from single-molecule mobility measurements with fluorescence correlation spectroscopy to spatio-temporal modulation of resource sharing in cell-free gene expression bursting.

The three-dimensional (3D) culture of neural cells in extracellular matrix (ECM) gels holds promise for modeling neurodegenerative diseases and pre-clinical evaluation of novel therapeutics. However, most current strategies for fabricating 3D neural cell cultures are not well suited to automated production and analysis. Here, we present a facile, replicable, 3D cell culture system that is compatible with standard laboratory equipment and high-throughput workflows. This system uses aqueous two-phase systems (ATPSs) to confine small volumes (5 and 10 μl) of a commonly used ECM hydrogel (Matrigel) into thin, discrete layers, enabling highly-uniform production of 3D neural cell cultures in a 96-well plate format. These 3D neural cell cultures can be readily analyzed by epifluorescence microscopy and microplate reader. Our preliminary results show that many common polymers used in ATPSs interfere with Matrigel gelation and instead form fibrous precipitates. However, 0.5% hydroxypropyl methylcellulose (HPMC) and 2.5% dextran 10 kDa (D10) were observed to retain Matrigel integrity and had minimal impact on cell viability. This novel system offers a promising yet accessible platform for high-throughput fabrication of 3D neural tissues using readily available and cost-effective materials.

Extrusion-based bioprinting (EBP) is limited by loss of pattern fidelity when printing on wet substrates. This can be overcome using aqueous two-phase systems (ATPSs) as novel ink formulations for EBP. In this study, optimal concentrations of ATPS “inks” were determined and used to pattern human epidermal keratinocyte (HEK001) colonies on a wet substrate for promoting epidermal growth. Four equilibrated and non-equilibrated ATPS formulations were tested for stable ATPS formation and uniform cell patterning. We identified an optimal formulation that produced stable droplets on a standard tissue culture plate coated with PEG. This process was also tested on an acellular dermal matrix (DermGENTM ) to evaluate biopattern fidelity on a tissue matrix. Cell proliferation and formation of adherens junctions between cells were analyzed by immunocytochemistry. Non-equilibrated 5.0% PEG and 5.0% DEX solutions formed tighter colonies than equilibrated solutions containing identical total polymer concentrations. Cells patterned in colonies displayed higher cell viability and increased formation of E-cadherin junctions compared to non-patterned cells. Finally, when the cells were patterned on DermGENTM , discrete cell colonies were observed. This suggests that ATPS EBP holds promise for biopatterning epidermal keratinocyte cells to improve skin tissue engineering.

Accurate analysis of microliter blood samples can improve patient care during medical testing and forensics. Patients can suffer from anemia due to the larger volume required for blood tests, 7 milliliters per vial. Attempts at analysis of nanoliter blood samples by Theranos have systematic errors > 10%, higher than medically acceptable thresholds. Our research aims to analyze composition of microliters of blood. This research investigates accuracy of analyzing blood via HemaDrop™, a patented technique to create a Homogenous Thin Solid Film (HTSF) on super-hydrophilic and hyper-hydrophilic surfaces with 5 microliter droplets of blood. To investigate HemaDrop™’s accuracy, Ion Beam Analysis (IBA) is conducted on dried blood spots (DBS) and HTSFs from congealed blood drops on HemaDrop™-treated samples. HTSFs are observed via optical microscopy to compare uniformity, precipitation, and phase separation. DBSs and HTSFs are compared via optical microscopy for canine blood and human blood. After drying uncoated samples, canine and human blood DBSs exhibit cratering, phase separation, and lack of uniformity. Conversely, HTSFs are uniform, exhibiting no cratering and little phase separation. Next, IBA demonstrates that HTSFs of canine and human blood solidified on super-hydrophilic and hyper-hydrophilic coatings yield spectra where species and electrolytes can be identified, unlike on DBSs. The damage curve method enables extracting accurate blood composition for elements, accounting for IBA damage. Relative error in blood elemental composition is within the 10% medical threshold. While both produced films within the 10% threshold, hyper-hydrophilic coatings eliminated phase separation from serum observed in HTSFs on super-hydrophilic coatings. HemaDrop™ provides consistent measurements independent of sample, showing HTSFs from µL blood drops are uniform, reproducible, and free of phase separation. Thus, HemaDrop™ allows for analysis in vacuum from congealed blood drops and expands the range of techniques to identify elements and molecules.