Joseph M. Labuz

High-yield isolation of extracellular vesicles using aqueous two-phase system.

Extracellular vesicles (EVs) such as exosomes and microvesicles released from cells are potential biomarkers for blood-based diagnostic applications. To exploit EVs as diagnostic biomarkers, an effective pre-analytical process is necessary. However, recent studies performed with blood-borne EVs have been hindered by the lack of effective purification strategies. In this study, an efficient EV isolation method was developed by using polyethylene glycol/dextran aqueous two phase system (ATPS). This method provides high EV recovery efficiency (~70%) in a short time (~15 min). Consequently, it can significantly increase the diagnostic applicability of EVs.

Pharmacokinetic profile that reduces nephrotoxicity of gentamicin in a perfused kidney-on-a-chip.

Nephrotoxicity is often underestimated because renal clearance in animals is higher compared to in humans. This paper aims to illustrate the potential to fill in such pharmacokinetic gaps between animals and humans using a microfluidic kidney model. As an initial demonstration, we compare nephrotoxicity of a drug, administered at the same total dosage, but using different pharmacokinetic regimens. Kidney epithelial cell, cultured under physiological shear stress conditions, are exposed to gentamicin using regimens that mimic the pharmacokinetics of bolus injection or continuous infusion in humans. The perfusion culture utilized is important both for controlling drug exposure and for providing cells with physiological shear stress (1.0 dyn cm(-2)). Compared to static cultures, perfusion culture improves epithelial barrier function. We tested two drug treatment regimens that give the same gentamycin dose over a 24 h period. In one regimen, we mimicked drug clearance profiles for human bolus injection by starting cell exposure at 19.2 mM of gentamicin and reducing the dosage level by half every 2 h over a 24 h period. In the other regimen, we continuously infused gentamicin (3 mM for 24 h). Although junctional protein immunoreactivity was decreased with both regimens, ZO-1 and occludin fluorescence decreased less with the bolus injection mimicking regimen. The bolus injection mimicking regimen also led to less cytotoxicity and allowed the epithelium to maintain low permeability, while continuous infusion led to an increase in cytotoxicity and permeability. These data show that gentamicin disrupts cell-cell junctions, increases membrane permeability, and decreases cell viability particularly with prolonged low-level exposure. Importantly a bolus injection mimicking regimen alleviates much of the nephrotoxicity compared to the continuous infused regimen. In addition to potential relevance to clinical gentamicin administration regimens, the results are important in demonstrating the general potential of using microfluidic cell culture models for pharmacokinetics and toxicity studies.

Designing 3-D adipospheres for quantitative metabolic study

Quantitative assessment of adipose mitochondrial activity is critical for better understanding of adipose tissue function in obesity and diabetes. While the two-dimensional (2-D) tissue culture method has been sufficient to discover key molecules that regulate adipocyte differentiation and function, the method is insufficient to determine the role of extracellular matrix (ECM) molecules and their modifiers, such as matrix metalloproteinases (MMPs), in regulating adipocyte function in three-dimensional (3-D) in vivo-like microenvironments. By using a 3-D hanging drop tissue culture system, we are able to produce scalable 3-D adipospheres that are suitable for quantitative metabolic study in 3-D microenvironment.

Bioprinting using aqueous two-phase system

A key for successful bioprinting is the design and formulation of a suitable “ink” that maintains cell viability while being patternable. Here, we describe the uses of aqueous two-phase systems (ATPS) to bioprint or micropattern cells as well as reagents. The unique advantage of the method compared to other bioprinting methods is that one can pattern while fully immersed in aqueous solutions without diffusion or dispersion of the aqueous ink. The fully aqueous environment is advantageous for cell printing where even brief drying can be lethal. Additionally, bioprinting with ATPS is typically performed in a noncontact manner allowing printing over delicate materials such as living cells, tissues, and hydrogels straightforward. While bioprinting generally implies additive fabrication, sculpting of existing biological structures can also serve to create cellular patterns. This chapter thus provides an overview of the use of ATPS bioinks to perform both additive and subtractive fabrication.

Organs-on-a-Chip

Organs-on-a-chip are microfluidic cell and tissue culture platforms capable of recapitulating key physiologies and functions of specific organs for further biological and medical purposes. A considerable number of such devices have been developed for many different organs such as blood vessels, lung, and liver. The development of organ-on-a-chip devices has helped widen our understanding of biological phenomena. Beyond basic science, organ-on-a-chip devices have also contributed to pharmacological studies. Despite the many outstanding achievements of previous organ-on-a-chip studies, there is still room for further improvement such as the improved modeling of fundamental organ processes as well as integration of individual organ modules into a generalizable human-on-a-chip.