In situ, dual-mode monitoring of organ-on-a-chip with smartphone-based fluorescence microscope

Highlights

• Non-destructive, in situ monitoring of drug-induced nephrotoxicity on kidney-on-a-chip.

• Dual mode monitoring of both inside and outside the organ-on-a-chip.

• Immunocapture and immunoagglutination monitor both on-chip expression and outflow shedding.

• Smartphone-based fluorescence microscope quantifies immunocapture and immunoagglutination.

Abstract

The use of organ-on-a-chip (OOC) platforms enables improved simulation of the human kidney's response to nephrotoxic drugs. The standard method of analyzing nephrotoxicity from existing OOC has majorly consisted of invasively collecting samples (cells, lysates, media, etc.) from an OOC. Such disruptive analyses potentiate contamination, disrupt the replicated in vivo environment, and require expertize to execute. Moreover, traditional analyses, including immunofluorescence microscopy, immunoblot, and microplate immunoassay are essentially not in situ and require substantial time, resources, and costs. In the present work, the incorporation of fluorescence nanoparticle immunocapture/immunoagglutination assay into an OOC enabled dual-mode monitoring of drug-induced nephrotoxicity in situ. A smartphone-based fluorescence microscope was fabricated as a handheld in situ monitoring device attached to an OOC. Both the presence of γ-glutamyl transpeptidase (GGT) on the apical brush-border membrane of 786-O proximal tubule cells within the OOC surface, and the release of GGT to the outflow of the OOC were evaluated with the fluorescence scatter detection of captured and immunoagglutinated anti-GGT conjugated nanoparticles. This dual-mode assay method provides a novel groundbreaking tool to enable the internal and external in situ monitoring of the OOC, which may be integrated into any existing OOCs to facilitate their subsequent analyses.

Introduction

Organ-on-a-chip (OOC) is defined as an integration of advanced 3D tissue engineered constructs with microfluidic network systems, i.e., lab-on-a-chip (LOC) (Bhise et al., 2014). Human cells are seeded and proliferated within the LOCs, providing a realistic replicate of the human organ. Such novel platforms improve preclinical testing of drugs, implants, biomedical devices, or stem cell therapies, due to their more realistic simulation of physiological function. OOCs have been developed to mimic the functions of various organs, including kidney, liver, brain, heart, skeletal muscle, and intestine (Ghaemmaghami et al., 2012, Esch et al., 2012). In OOCs, fluid flow generates mechanical forces that recapitulate the in vivo microenvironment experienced by cells, which cannot be accomplished by static 2D mammalian cell culture (Bhise et al., 2014). In this work, we are interested in developing kidney-on-a-chip to assess chemical-induced toxicity. Kidney is the main excretory organ that is exposed to drugs and xenobiotics. The epithelial cells of the renal proximal tubules in kidney are the most susceptible target for such drugs and xenobiotics (referred to as nephrotoxicants), due to their roles in the concentration of glomerular filtrate and their capacity for drug metabolism (Tiong et al., 2014). Specifically, previous studies of kidney-on-a-chip observed that proximal tubular cell (PTCs) functionality and morphology are improved under flow conditions (Jang et al., 2013), which is critical to emulate a response similar to the human kidney.

However, assessment of cell response during such OOC experiments remain tedious, disruptive, time-consuming, and lack real-time in situ analyses (Cai et al., 2015, Jang et al., 2013, Johnson et al., 2016, Maschmeyer et al., 2015). For example, a reported kidney-on-a-chip required immunofluorescence microscopy of the membrane within the OOC via cell fixation to monitor the growth of the epithelial cells (thus not in situ) and immunoblot of cells harvested from the OOC post-treatment (Jang et al., 2013). Moreover, both methods require overnight analyses steps and invasive collection of the membrane and cells. Consequentially, the irreversible, permanent bonding of polydimethyl siloxane (PDMS)-based OOCs, the most common material used for not only OOCs but also bulk of LOCs, challenges the users in accessing, isolating, and processing cultured cells for certain preferred analysis including histology and electron microscopy (Huh et al., 2013). Thus, the users are subject to destructive methods of subsequent analyses of cells, or may heavily rely on fluorescence microscopy after fixation and subsequent immunostaining (Huh et al., 2012, Huh et al., 2013, van der Meer et al., 2013, Maschmeyer et al., 2015, Johnson et al., 2016). In several studies, the secreted cellular products have been analyzed, but they generally lacked the required sensitivity due to the low cell count (~10,000 cells) and the dilution of such products upon continuous perfusion (Huh et al., 2013). Therefore, existing OOCs can be significantly improved by application of a highly sensitive, direct detection tool to assess cytotoxicity, e.g., immediate and in situ quantification of the changes in target cell/protein concentrations. It is also preferable to fabricate such a tool as simply and inexpensively as possible to ensure ready availability to the widest selection of researchers.

In order to demonstrate our in situ, dual-mode monitoring tool, we propose to prototype a simple OOC with the use of 3D printing and a common house-use cutter machine. We will use a 3D printed template for conventional soft lithography towards fabrication of PDMS-based OOC (Comina et al., 2014), as a simpler and faster alternative. Since 3D printing does not provide sufficient resolution required for promoting cellular adhesion on its surface, we will use a common house-use cutter machine to create the patterns on the inner channel surfaces of an OOC. Although a cutter machine has been previously utilized to “cut” specific channel layouts to fabricate paper-based LOC devices (Fang et al., 2015, Fang et al., 2014), it has not yet been utilized to modify the surface topography of LOC or OOC devices. This technique can provide a simpler and affordable alternative for adding micro-scale structures to the 3D printed LOC or OOC devices. Such textural detail addition may improve cellular adhesion to the substrates of OOCs.

Most importantly, a non-invasive, in situ monitoring tool needs to be incorporated into OOC, which should be easy to use, affordable, and potentially handheld, yet provide accurate and specific assay results. With this goal in mind, we propose the use of a fluorescent nanoparticle immunocapture as well as immunoagglutination assay coupled to a smartphone-based fluorescence microscope. This method will ultimately reduce assay time, offer sufficient assay specificity, ease of fabrication and use, while drastically reducing costly analytical procedures for in situ monitoring of cytotoxicity on OOC. Renal proximal tubule derived cells (PTCs) express various PTC-specific brush border enzymes, such as γ-glutamyl transpeptidase (GGT), a protein that catalyzes the first step in the metabolism of glutathione (GSH) and GSH conjugates (Tiong et al., 2014). In response to PTC toxicants, the brush border membrane frequently sheds, releasing GGT into the tubular lumen, providing a desired target for detection of cytotoxicity. The use of nanoparticle immunocapture/immunoagglutination for in situ monitoring can be incorporated into any existing OOC system by altering the antibody to any given target. Thus, researchers will benefit greatly by improving their OOC analysis techniques. Our report represents the first demonstration of incorporating particle immunocapture/immunoagglutination assays to OOC systems.

Our group has quantified the concentration of bacteria by evaluating the angle-specific Mie scatter signal from immunoagglutinated polystyrene particles (You et al., 2011, Fronczek et al., 2013, Park et al., 2013, Cho et al., 2015). Using smartphone-based optical detection, Escherichia coli and Neisseria gonorrhoeae were sensitively and specifically detected from undiluted human urine, a complicated bodily fluid (Cho et al., 2015). Particle concentration was optimized to detect a varying range of pathogen concentration. Our group has also previously constructed a 3D printed smartphone-based fluorescence microscope for the end-point quantification of PCR products (Angus et al., 2015). As emphasized, existing OOCs and LOCs would benefit greatly from a versatile analysis method that is noninvasive to the device and its content. With the intent of performing analysis on-chip, many approaches have been developed, including electrochemical electrodes, optical sensors, label-free detection of molecules, field-effect transistor sensors, and micro-cantilevers (Sung et al., 2013). One such proof-of-concept study was a portable fluorescence optical detection system to analyze the dynamics of cell viability (Choi et al., 2010). However, the detailed optical system is quite technical and a static 3D culture was fluorescently dyed, which would limit the scope of its application. Cells ideally need to be exposed under flow, especially when studying the cytotoxic response of cells that line directional flow with fluid-filled compartments, which is a similar microenvironment as human tissue.

In the current work, we predicted that the anti-GGT conjugated nanoparticles would be immunoagglutinated upon binding with the GGT either specifically released from the damaged PTCs within the OOC, or captured on the membrane fragments also released from the brush border membrane of PTCs (Fig. 1). Both behaviors can be monitored in situ through the use of fluorescent nanoparticles and subsequent detection via a smartphone-based fluorescence microscope. The method provides a novel groundbreaking tool, enabling in situ, dual-mode monitoring of the internal and external compartments of the OOC, and which may be integrated into any existing OOCs to facilitate analyses. This dual-mode of detection, i.e., nanoparticle immunoagglutination and particle capture, has not been previously demonstrated, which is particularly suited for OOC experiments in monitoring both membrane expression and subsequent release of protein products, without the need for collecting, fixing, and/or staining the cells.