Organ-level physiology is recapitulated in vitro by culturing cells in perfused, microfluidic devices.

Microfluidic organs-on-chips

This critical review summarizes developments in microfluidic platforms that enable the miniaturization, integration, automation and parallelization of (bio-)chemical assays (see S. Haeberle and R. Zengerle, Lab Chip, 2007, 7, 1094–1110, for an earlier review). In contrast to isolated application-specific solutions, a microfluidic platform provides a set of fluidic unit operations, which are designed for easy combination within a well-defined fabrication technology. This allows the easy, fast, and cost-efficient implementation of different application-specific (bio-)chemical processes. In our review we focus on recent developments from the last decade (2000s). We start with a brief introduction into technical advances, major market segments and promising applications. We continue with a detailed characterization of different microfluidic platforms, comprising a short definition, the functional principle, microfluidic unit operations, application examples as well as strengths and limitations of every platform. The microfluidic platforms in focus are lateral flow tests, linear actuated devices, pressure driven laminar flow, microfluidic large scale integration, segmented flow microfluidics, centrifugal microfluidics, electrokinetics, electrowetting, surface acoustic waves, and dedicated systems for massively parallel analysis. This review concludes with the attempt to provide a selection scheme for microfluidic platforms which is based on their characteristics according to key requirements of different applications and market segments. Applied selection criteria comprise portability, costs of instrument and disposability, sample throughput, number of parameters per sample, reagent consumption, precision, diversity of microfluidic unit operations and the flexibility in programming different liquid handling protocols (295 references).

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Microfluidic lab-on-a-chip platforms: requirements, characteristics and applications

Transepithelial/transendothelial electrical resistance (TEER) is a widely accepted quantitative technique to measure the integrity of tight junction dynamics in cell culture models of endothelial and epithelial monolayers. TEER values are strong indicators of the integrity of the cellular barriers before they are evaluated for transport of drugs or chemicals. TEER measurements can be performed in real time without cell damage and generally are based on measuring ohmic resistance or measuring impedance across a wide spectrum of frequencies. The measurements for various cell types have been reported with commercially available measurement systems and also with custom-built microfluidic implementations. Some of the barrier models that have been widely characterized using TEER include the blood–brain barrier (BBB), gastrointestinal (GI) tract, and pulmonary models. Variations in these values can arise due to factors such as temperature, medium formulation, and passage number of cells. The aim of this article ...

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TEER Measurement Techniques for In Vitro Barrier Model Systems

Microengineered cell culture systems are becoming sufficiently sophisticated that they can recapitulate many of the phenomena observed in tissues and organisms. Here, Huh and colleagues discuss the advances made in these 'organs-on-chips' and how they could be used in drug development, including target identification and validation, toxicity screening and stratified medicine.

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Organs-on-chips at the frontiers of drug discovery

Cell fusion has been used for many different purposes, including generation of hybridomas and reprogramming of somatic cells. The fusion step is the key event in initiation of these procedures. Standard fusion techniques, however, provide poor and random cell contact, leading to low yields. We present here a microfluidic device to trap and properly pair thousands of cells. Using this device, we paired different cell types, including fibroblasts, mouse embryonic stem cells and myeloma cells, achieving pairing efficiencies up to 70%. The device is compatible with both chemical and electrical fusion protocols. We observed that electrical fusion was more efficient than chemical fusion, with membrane reorganization efficiencies of up to 89%. We achieved greater than 50% properly paired and fused cells over the entire device, fivefold greater than with a commercial electrofusion chamber and observed reprogramming in hybrids between mouse embryonic stem cells and mouse embryonic fibroblasts.

Microfluidic control of cell pairing and fusion.

The blood-brain barrier (BBB) is a unique feature of the human body, preserving brain homeostasis and preventing toxic substances to enter the brain. However, in various neurodegenerative diseases, the function of the BBB is disturbed. Mechanisms of the breakdown of the BBB are incompletely understood and therefore a realistic model of the BBB is essential. We present here the smallest model of the BBB yet, using a microfluidic chip, and the immortalized human brain endothelial cell line hCMEC/D3. Barrier function is modulated both mechanically, by exposure to fluid shear stress, and biochemically, by stimulation with tumor necrosis factor alpha (TNF-α), in one single device. The device has integrated electrodes to analyze barrier tightness by measuring the transendothelial electrical resistance (TEER). We demonstrate that hCMEC/D3 cells could be cultured in the microfluidic device up to 7 days, and that these cultures showed comparable TEER values with the well-established Transwell assay, with an average (± SEM) of 36.9 Ω.cm2 (± 0.9 Ω.cm2) and 28.2 Ω.cm2 (± 1.3 Ω.cm2) respectively. Moreover, hCMEC/D3 cells on chip expressed the tight junction protein Zonula Occludens-1 (ZO-1) at day 4. Furthermore, shear stress positively influenced barrier tightness and increased TEER values with a factor 3, up to 120 Ω.cm2. Subsequent addition of TNF-α decreased the TEER with a factor of 10, down to 12 Ω.cm2. This realistic microfluidic platform of the BBB is very well suited to study barrier function in detail and evaluate drug passage to finally gain more insight into the treatment of neurodegenerative diseases.

BBB ON CHIP: microfluidic platform to mechanically and biochemically modulate blood-brain barrier function

There is great interest in genetic modification of bone marrow-derived mesenchymal stem cells (MSC), not only for research purposes but also for use in (autologous) patient-derived-patient-used transplantations. A major drawback of bulk methods for genetic modifications of (stem) cells, like bulk-electroporation, is its limited yield of DNA transfection (typically then 10%). This is even more limited when cells are present at very low numbers, as is the case for stem cells. Here we present an alternative technology to transfect cells with high efficiency (>75%), based on single cell electroporation in a microfluidic device. In a first experiment we show that we can successfully transport propidium iodide (PI) into single mouse myoblastic C2C12 cells. Subsequently, we show the use of this microfluidic device to perform successful electroporation of single mouse myoblastic C2C12 cells and single human MSC with vector DNA encoding a green fluorescent-erk1 fusion protein (EGFP-ERK1 (MAPK3)). Finally, we performed electroporation in combination with live imaging of protein expression and dynamics in response to extracellular stimuli, by fibroblast growth factor (FGF-2). We observed nuclear translocation of EGFP-ERK1 in both cell types within 15 min after FGF-2 stimulation. Due to the successful and promising results, we predict that microfluidic devices can be used for highly efficient small-scale genetic modification of cells, and biological experimentation, offering possibilities to study cellular processes at the single cell level. Future applications might be small-scale production of cells for therapeutic application under controlled conditions.

P.M. ter Braak

Papers

BBB ON CHIP: microfluidic platform to mechanically and biochemically modulate blood-brain barrier function

Gene transfer and protein dynamics in stem cells using single cell electroporation in a microfluidic device