From 3D cell culture to organs-on-chips.

Where, When, and How: Context-Dependent Functions of RNA Methylation Writers, Readers, and Erasers

'Organs-on-chips' are microengineered biomimetic systems containing microfluidic channels lined by living human cells, which replicate key functional units of living organs to reconstitute integrated human organ-level pathophysiology in vitro. These microdevices can be used to test efficacy and toxicity of drugs and chemicals, and to create in vitro models of human disease. Thus, they potentially represent low-cost alternatives to conventional animal models for pharmaceutical, chemical and environmental applications. Here we describe a protocol for the fabrication, microengineering and operation of these microfluidic organ-on-chip systems. First, microengineering is used to fabricate a multilayered microfluidic device that contains two parallel elastomeric microchannels separated by a thin porous flexible membrane, along with two full-height, hollow vacuum chambers on either side; this requires ∼3.5 d to complete. To create a 'breathing' lung-on-a-chip that mimics the mechanically active alveolar-capillary interface of the living human lung, human alveolar epithelial cells and microvascular endothelial cells are cultured in the microdevice with physiological flow and cyclic suction applied to the side chambers to reproduce rhythmic breathing movements. We describe how this protocol can be easily adapted to develop other human organ chips, such as a gut-on-a-chip lined by human intestinal epithelial cells that experiences peristalsis-like motions and trickling fluid flow. Also, we discuss experimental techniques that can be used to analyze the cells in these organ-on-chip devices.

Microfabrication of human organs-on-chips

Since the identification of the first RNA demethylase and the establishment of methylated RNA immunoprecipitation-sequencing methodology 6 to 7 years ago, RNA methylation has emerged as a widespread phenomenon and a critical regulator of transcript expression. This new layer of regulation is termed “epitranscriptomics.” The most prevalent RNA methylation, N6-methyladenosine (m6A), occurs in approximately 25% of transcripts at the genome-wide level and is enriched around stop codons, in 5′- and 3′-untranslated regions, and within long internal exons. RNA m6A modification regulates RNA splicing, translocation, stability, and translation into protein. m6A is catalyzed by the RNA methyltransferases METTL3, METTL14, and METTL16 (writers), is removed by the demethylases FTO and ALKBH5 (erasers), and interacts with m6A-binding proteins, such as YTHDF1 and IGF2BP1 (readers). RNA methyltransferases, demethylases, and m6A-binding proteins are frequently upregulated in human cancer tissues from a variety of organ origins, increasing onco-transcript and oncoprotein expression, cancer cell proliferation, survival, tumor initiation, progression, and metastasis. Although RNA methyltransferase inhibitors are not available yet, FTO inhibitors have shown promising anticancer effects in vitro and in animal models of cancer. Further screening for selective and potent RNA methyltransferase, demethylase, or m6A-binding protein inhibitors may lead to compounds suitable for future clinical trials in cancer patients.

https://cancerres.aacrjournals.org/content/canres/79/7/1285.full.pdf

The Critical Role of RNA m6A Methylation in Cancer

The ultimate goal of most biomedical research is to gain greater insight into mechanisms of human disease or to develop new and improved therapies or diagnostics. Although great advances have been made in terms of developing disease models in animals, such as transgenic mice, many of these models fail to faithfully recapitulate the human condition. In addition, it is difficult to identify critical cellular and molecular contributors to disease or to vary them independently in whole-animal models. This challenge has attracted the interest of engineers, who have begun to collaborate with biologists to leverage recent advances in tissue engineering and microfabrication to develop novel in vitro models of disease. As these models are synthetic systems, specific molecular factors and individual cell types, including parenchymal cells, vascular cells, and immune cells, can be varied independently while simultaneously measuring system-level responses in real time. In this article, we provide some examples of these efforts, including engineered models of diseases of the heart, lung, intestine, liver, kidney, cartilage, skin and vascular, endocrine, musculoskeletal, and nervous systems, as well as models of infectious diseases and cancer. We also describe how engineered in vitro models can be combined with human inducible pluripotent stem cells to enable new insights into a broad variety of disease mechanisms, as well as provide a test bed for screening new therapies.

Engineered in vitro disease models.

N6-methyladenosine (m6A) is a reversible modification in mRNA and has been shown to regulate processing, translation and decay of mRNA. However, the roles of m6A modification in neuronal development are still not known. Here, we found that the m6A eraser FTO is enriched in axons and can be locally translated. Axon-specific inhibition of FTO by rhein, or compartmentalized siRNA knockdown of Fto in axons led to increases of m6A levels. GAP-43 mRNA is modified by m6A and is a substrate of FTO in axons. Loss-of-function of this non-nuclear pool of FTO resulted in increased m6A modification and decreased local translation of axonal GAP-43 mRNA, which eventually repressed axon elongation. Mutation of a predicted m6A site in GAP-43 mRNA eliminated its m6A modification and exempted regulation of its local translation by axonal FTO. This work showed an example of dynamic internal m6A demethylation of non-nuclear localized mRNA by the demethylase FTO. Regulation of m6A modification of axonal mRNA by axonal FTO might be a general mechanism to control their local translation in neuronal development.

/pdf/dynamic-m6a-modification-regulates-local-translation-of-mrna-q1etjfb94h.pdf

Dynamic m6A modification regulates local translation of mRNA in axons.

This paper presents a circular microfluidic compartmentalized co-culture platform that can be used for central nervous system (CNS) axon myelination research. The microfluidic platform is composed of a soma compartment and an axon/glia compartment connected through arrays of axon-guiding microchannels. Myelin-producing glia, oligodendrocytes (OLs), placed in the axon/glia compartment, interact with only axons but not with neuronal somata confined to the soma compartment, reminiscent to in vivo situation where many axon fibres are myelinated by OLs at distance away from neuronal cell bodies. Primary forebrain neurons from embryonic day 16-18 rats were cultured inside the soma compartment for two weeks to allow them to mature and form extensive axon networks. OL progenitors, isolated from postnatal day 1-2 rat brains, were then added to the axon/glia compartment and co-cultured with neurons for an additional two weeks. The microdevice showed fluidic isolation between the two compartments and successfully isolated neuronal cell bodies and dendrites from axons growing through the arrays of axon-guiding microchannels into the axon/glia compartment. The circular co-culture device developed here showed excellent cell loading characteristics where significant numbers of cells were positioned near the axon-guiding microchannels. This significantly increased the probability of axons crossing these microchannels as demonstrated by the more than 51 % of the area of the axon/glia compartment covered with axons two weeks after cell seeding. OL progenitors co-cultured with axons inside the axon/glia compartment successfully differentiated into mature OLs. These results indicate that this device can be used as an excellent in vitro co-culture platform for studying localized axon-glia interaction and signalling.

Microfluidic compartmentalized co-culture platform for CNS axon myelination research.

Objective: The blood–brain barrier (BBB) poses a unique challenge to the development of therapeutics against neurological disorders due to its impermeabi-lity to most of the chemical compounds. Most in vitro BBB models have limitations in mimicking in vivo conditions and functions. Here, we show a co-culture microfluidic BBB-on-a-chip that provides interactions between neurovascular endothelial cells and neuronal cells across a porous polycarbonate membrane, which better mimics the in vivo conditions, as well as allows in vivo level shear stress to be applied. Methods: A 4 × 4 intersecting microchannel array forms 16 BBB sites on a chip, with a multielectrode array integrated to measure the transendothelial electrical resistance (TEER) from all 16 different sites, which allows label-free real-time analysis of the barrier function. Primary mouse endothelial cells and primary astrocytes were co-cultured in the chip while applying in vivo level shear stress. The chip allows the barrier function to be analyzed through TEER measurement, dextran permeability, as well as immunostaining. Results: Co-culture between astrocytes and endothelial cells, as well as in vivo level shear stress applied, led to the formation of tighter junctions and significantly lower barrier permeability. Moreover, drug testing with histamine showed increased permeability when using only endothelial cells compared to almost no change when using co-culture. Conclusion: Results show that the developed BBB chip more closely mimics the in vivo BBB environment. Significance: The developed multisite BBB chip is expected to be used for screening drug by more accurately predicting their permeability through BBB as well as their toxicity.

A Three-Dimensional Arrayed Microfluidic Blood–Brain Barrier Model With Integrated Electrical Sensor Array

Formation of myelin sheaths by oligodendrocytes (OLs) in the central nervous system (CNS) is essential for rapid nerve impulse conduction. Reciprocal signaling between axons and OLs orchestrates myelinogenesis but remains largely elusive. In this study, we present a multi-compartment CNS neuron–glia microfluidic co-culture platform. The platform is capable of conducting parallel localized drug and biomolecule treatments while carrying out multiple co-culture conditions in a single device for studying axon–glia interactions at a higher throughput. The “micro-macro hybrid soft-lithography master fabrication” (MMHSM) technique enables a large number of precisely replicated PDMS devices incorporating both millimeter and micrometer scale structures to be rapidly fabricated without any manual reservoir punching processes. Axons grown from the neuronal somata were physically and fluidically isolated inside the six satellite axon/glia compartments for localized treatments. Astrocytes, when seeded and co-cultured after the establishment of the isolated axons in the satellite axon/glia compartments, were found to physically damage the established axonal layer, as they tend to grow underneath the axons. In contrast, oligodendrocyte progenitor cells (OPCs) could be co-cultured successfully with the isolated axons and differentiated into mature myelin basic protein-expressing OLs with processes aligning to neighboring axons. OPCs inside the six axon/glia compartments were treated with a high concentration of ceramide (150 μM) to confirm the fluidic isolation among the satellite compartments. In addition, isolated axons were treated with varying concentrations of chondroitin sulfate proteoglycan (CSPG, 0–25 μg ml−1) within a single device to demonstrate the parallel localized biomolecular treatment capability of the device. These results indicate that the proposed platform can be used as a powerful tool to study CNS axonal biology and axon–glia interactions with the capacity for localized biomolecular treatments.

/pdf/multi-compartment-neuron-glia-co-culture-platform-for-1td9u20isz.pdf

Jaewon Park

Papers

Dynamic m6A modification regulates local translation of mRNA in axons.

Microfluidic compartmentalized co-culture platform for CNS axon myelination research.

A Three-Dimensional Arrayed Microfluidic Blood–Brain Barrier Model With Integrated Electrical Sensor Array

Multi-Compartment Neuron-Glia Co-culture Platform for Localized CNS Axon-glia Interaction Study

A microchip for quantitative analysis of CNS axon growth under localized biomolecular treatments.