Patrick M. Misun

Multi-analyte biosensor interface for real-time monitoring of 3D microtissue spheroids in hanging-drop networks

Microfluidics is becoming a technology of growing interest for building microphysiological systems with integrated read-out functionalities. Here we present the integration of enzyme-based multi-analyte biosensors into a multi-tissue culture platform for ‘body-on-a-chip’ applications. The microfluidic platform is based on the technology of hanging-drop networks, which is designed for the formation, cultivation, and analysis of fluidically interconnected organotypic spherical three-dimensional (3D) microtissues of multiple cell types. The sensor modules were designed as small glass plug-ins featuring four platinum working electrodes, a platinum counter electrode, and an Ag/AgCl reference electrode. They were placed directly into the ceiling substrate from which the hanging drops that host the spheroid cultures are suspended. The electrodes were functionalized with oxidase enzymes to enable continuous monitoring of lactate and glucose through amperometry. The biosensors featured high sensitivities of 322±41 nA mM−1 mm−2 for glucose and 443±37 nA mM−1 mm−2 for lactate; the corresponding limits of detection were below 10 μM. The proposed technology enabled tissue-size-dependent, real-time detection of lactate secretion from single human colon cancer microtissues cultured in the hanging drops. Furthermore, glucose consumption and lactate secretion were monitored in parallel, and the impact of different culture conditions on the metabolism of cancer microtissues was recorded in real-time.

Seamless Combination of Fluorescence-Activated Cell Sorting and Hanging-Drop Networks for Individual Handling and Culturing of Stem Cells and Microtissue Spheroids

Open microfluidic cell culturing devices offer new possibilities to simplify loading, culturing, and harvesting of individual cells or microtissues due to the fact that liquids and cells/microtissues are directly accessible. We present a complete workflow for microfluidic handling and culturing of individual cells and microtissue spheroids, which is based on the hanging-drop network concept: The open microfluidic devices are seamlessly combined with fluorescence-activated cell sorting (FACS), so that individual cells, including stem cells, can be directly sorted into specified culturing compartments in a fully automated way and at high accuracy. Moreover, already assembled microtissue spheroids can be loaded into the microfluidic structures by using a conventional pipet. Cell and microtissue culturing is then performed in hanging drops under controlled perfusion. On-chip drop size control measures were applied to stabilize the system. Cells and microtissue spheroids can be retrieved from the chip by using a parallelized transfer method. The presented methodology holds great promise for combinatorial screening of stem-cell and multicellular-spheroid cultures.

Smart Cell Culture Systems: Integration of Sensors and Actuators into Microphysiological Systems

Technological advances in microfabrication techniques in combination with organotypic cell and tissue models have enabled the realization of microphysiological systems capable of recapitulating aspects of human physiology in vitro with great fidelity. Concurrently, a number of analysis techniques has been developed to probe and characterize these model systems. However, many assays are still performed off-line, which severely compromises the possibility of obtaining real-time information from the samples under examination, and which also limits the use of these platforms in high-throughput analysis. In this review, we focus on sensing and actuation schemes that have already been established or offer great potential to provide in situ detection or manipulation of relevant cell or tissue samples in microphysiological platforms. We will first describe methods that can be integrated in a straightforward way and that offer potential multiplexing and/or parallelization of sensing and actuation functions. These methods include electrical impedance spectroscopy, electrochemical biosensors, and the use of surface acoustic waves for manipulation and analysis of cells, tissue, and multicellular organisms. In the second part, we will describe two sensor approaches based on surface-plasmon resonance and mechanical resonators that have recently provided new characterization features for biological samples, although technological limitations for use in high-throughput applications still exist.

Fabrication and Operation of Microfluidic Hanging-Drop Networks

The hanging-drop network (HDN) is a technology platform based on a completely open microfluidic network at the bottom of an inverted, surface-patterned substrate. The platform is predominantly used for the formation, culturing, and interaction of self-assembled spherical microtissues (spheroids) under precisely controlled flow conditions. Here, we describe design, fabrication, and operation of microfluidic hanging-drop networks.

Miniature Fluidic Microtissue Culturing Device for Rapid Biological Detection

Microfluidics is becoming a technology of growing interest to build miniature culturing systems, capable of mimicking tissue functions and multi-tissue interactions in so-called “body-on-a-chip ” applications while featuring integrated readout functionalities. This chapter presents a highly versatile, modular and scalable analytical platform technology, which combines microfluidic hanging-drop networks with multi-analyte biosensors for in situ monitoring of the metabolism of 3D microtissues. The microfluidic platform is based on the hanging -drop network technology, which has been designed for formation, cultivation, and analysis of fluidically interconnected organotypic spherical 3D microtissues that can be obtained from various different cell types. The sensor modules were designed as small glass plug-ins, which allow for convenient functionalization and calibration of the sensors and do not interfere with the microfluidic functions. They were placed in the ceiling substrate, from which the hanging drops that host the spheroid cultures were suspended. The detection of secreted lactate of single microtissue spheroids will be presented. Further, we will demonstrate that it is possible to monitor microtissue lactate secretion and glucose consumption in parallel.

Microfluidic Hydrogel Hanging-Drop Network for Long-Term Culturing of 3D Microtissues and Simultaneous High-Resolution Imaging

Three‐dimensional (3D) microtissues, cultured in microfluidic platforms, enable to study complex biological mechanisms that cannot be replicated in two‐dimensional cell cultures. Deeper insights can be obtained if these 3D culture systems are rendered compatible with high‐resolution time‐lapse imaging systems, which requires precise placement and immobilization of the specimen while ensuring high viability and functionality of the 3D cell constructs. This article presents a versatile microfluidic platform for long‐term culturing and analysis of 3D microtissues. The platform is compatible with time‐lapse high‐resolution confocal microscopy. Hanging hydrogel drops enable the precise placement and stable immobilization of the microtissues in the microfluidic chip. The chip includes perfusion capability to apply drugs, staining and clearing solutions. The features of the chip are demonstrated by studying (i) colon cancer microtissues to monitor tissue growth and cell death; on‐chip clearing was used to augment the penetration depth for endpoint imaging; (ii) primary human liver microtissues were exposed to cytochalasin D to observe its effect on the bile canaliculi. The results obtained with both sample types demonstrate the suitability of the system for investigating complex processes in organotypic 3D microtissues, down to single‐cell level, and for observation of physiologically relevant processes at subcellular scale.

Microfluidic hydrogel hanging-drop network for high-resolution microscopy of 3D microtissues

We present a microfluidic chip for the culturing of 3D microtissues under varying perfusion conditions. The microtissues are individually immobilized at the bottom of hanging hydrogel microdrops and are located at defined positions inside the culturing chambers of the microfluidic network. The precision in positioning and the stability of the microtissues over time renders the platform ideal for multi-position, high-resolution time-lapse imaging and techniques involving cell tracking. At the same time, fabrication and operation of the chip is simple and robust and does not require dedicated equipment.

Real-time multi-analyte online monitoring of 3d cell cultures by integrated enzyme-based biosensors in hanging drop networks

We present the integration of enzyme-based lactate and glucose biosensors into hanging-drop networks. Hangingdrop networks are an ideal microfluidic platform for formation, cultivation, and continuous and long-term observation of 3D microtissues. The implementation of biosensors enables in-situ online monitoring of the effects of different culturing conditions and compound dosages on the microtissues. A hybrid approach including glass sensor modules embedded into a microfluidic polydimethylsiloxane (PDMS)-based chip facilitates system integration. The biosensors enable real-time recording of lactate production and glucose consumption of human colon carcinoma spheroids.