Modular automated microfluidic cell culture platform reduces glycolytic stress in cerebral cortex organoids

Organ-on-a-chip systems combine microfluidics, cell biology, and tissue engineering to culture 3D organ-specific in vitro models that recapitulate the biology and physiology of their in vivo counterparts. Here, we have developed a multiplex platform that automates the culture of individual organoids in isolated microenvironments at user-defined media flow rates. Programmable workflows allow the use of multiple reagent reservoirs that may be applied to direct differentiation, study temporal variables, and grow cultures long term. Novel techniques in polydimethylsiloxane (PDMS) chip fabrication are described here that enable features on the upper and lower planes of a single PDMS substrate. RNA sequencing (RNA-seq) analysis of automated cerebral cortex organoid cultures shows benefits in reducing glycolytic and endoplasmic reticulum stress compared to conventional in vitro cell cultures

High-throughput full-length single-cell mRNA-seq of rare cells

<div><p>Single-cell characterization techniques, such as mRNA-seq, have been applied to a diverse range of applications in cancer biology, yielding great insight into mechanisms leading to therapy resistance and tumor clonality. While single-cell techniques can yield a wealth of information, a common bottleneck is the lack of throughput, with many current processing methods being limited to the analysis of small volumes of single cell suspensions with cell densities on the order of 10⁷ per mL. In this work, we present a high-throughput full-length mRNA-seq protocol incorporating a magnetic sifter and magnetic nanoparticle-antibody conjugates for rare cell enrichment, and Smart-seq2 chemistry for sequencing. We evaluate the efficiency and quality of this protocol with a simulated circulating tumor cell system, whereby non-small-cell lung cancer cell lines (NCI-H1650 and NCI-H1975) are spiked into whole blood, before being enriched for single-cell mRNA-seq by EpCAM-functionalized magnetic nanoparticles and the magnetic sifter. We obtain high efficiency (> 90%) capture and release of these simulated rare cells via the magnetic sifter, with reproducible transcriptome data. In addition, while mRNA-seq data is typically only used for gene expression analysis of transcriptomic data, we demonstrate the use of full-length mRNA-seq chemistries like Smart-seq2 to facilitate variant analysis of expressed genes. This enables the use of mRNA-seq data for differentiating cells in a heterogeneous population by both their phenotypic and variant profile. In a simulated heterogeneous mixture of circulating tumor cells in whole blood, we utilize this high-throughput protocol to differentiate these heterogeneous cells by both their phenotype (lung cancer versus white blood cells), and mutational profile (H1650 versus H1975 cells), in a single sequencing run. This high-throughput method can help facilitate single-cell analysis of rare cell populations, such as circulating tumor or endothelial cells, with demonstrably high-quality transcriptomic data.</p></div

Epithelial and WBC gene expression levels.

<p>Clear differences in <i>CD45</i> (WBC marker) and <i>EpCAM/KRT7/KRT8</i> (epithelial) genes are observed between the white blood cells and the H1650 cells.</p

Differentiating simulated CTC subpopulations by mutational analysis.

<p>H1975 and H1650 cells are spiked into blood, isolated by magnetic separation, and sequenced. By looking at the variants present in the cells, we are able to observe the same subpopulation mix as were originally spiked into the blood sample. A full list of the genes is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188510#pone.0188510.s003" target="_blank">S3 Table</a>, going from left to right.</p

Differentiating simulated CTC subpopulations by gene expression analysis.

<p>H1975 and H1650 cells are spiked into blood, isolated by magnetic separation, and analyzed. Their gene expression levels are very similar, and are consistent with prior results on the individual pure populations for both cell lines. Two distinct subpopulations are identified by hierarchical clustering among the isolated cells with p < 0.05, with one being a putative H1650 subpopulation, and the other being a putative H1975 subpopulation.</p

List of reagents used for Smart-seq2 and their respective vendors and catalog numbers.

<p>List of reagents used for Smart-seq2 and their respective vendors and catalog numbers.</p

Efficiencies of different steps in this method.

<p>(a) The sifter shows high capture efficiencies (> 90%) for 2 NSCLC cell lines tested (H1650 and H1975). Additionally, 2 sets of negative controls were also done with H1650 cells, with no non-specific capture observed. These negative controls are run as per the regular experiments, but with non-antibody functionalized magnetic nanoparticles (negative control for non-specific nanoparticle capture), and without the application of a magnet (negative control for non-magnetic capture). (b) The sifter also exhibits good release properties of captured cells and magnetic nanoparticles (89%). Optical images illustrate the effectiveness of elution from the magnetic sifter. The sifter surface post-elution appears as pristine as the surface of a brand new sifter. (c) FACS sort efficiencies vary with sort purity settings. 2 sort settings on the Sony SH-800 cell sorter are tested. Efficiencies of 50% and 29% are observed for the semi-purity and ultra-purity modes respectively. A reduced purity setting is required for higher yields. By following the Smart-seq2 protocol exactly, we observed successful cDNA synthesis in 51% of the wells.</p

Picroscope: low-cost system for simultaneous longitudinal biological imaging.

Simultaneous longitudinal imaging across multiple conditions and replicates has been crucial for scientific studies aiming to understand biological processes and disease. Yet, imaging systems capable of accomplishing these tasks are economically unattainable for most academic and teaching laboratories around the world. Here, we propose the Picroscope, which is the first low-cost system for simultaneous longitudinal biological imaging made primarily using off-the-shelf and 3D-printed materials. The Picroscope is compatible with standard 24-well cell culture plates and captures 3D z-stack image data. The Picroscope can be controlled remotely, allowing for automatic imaging with minimal intervention from the investigator. Here, we use this system in a range of applications. We gathered longitudinal whole organism image data for frogs, zebrafish, and planaria worms. We also gathered image data inside an incubator to observe 2D monolayers and 3D mammalian tissue culture models. Using this tool, we can measure the behavior of entire organisms or individual cells over long-time periods

Human-specific NOTCH2NL genes affect notch signaling and cortical neurogenesis

Genetic changes causing brain size expansion in human evolution have remained elusive. Notch signaling is essential for radial glia stem cell proliferation and is a determinant of neuronal number in the mammalian cortex. We find that three paralogs of human-specific NOTCH2NL are highly expressed in radial glia. Functional analysis reveals that different alleles of NOTCH2NL have varying potencies to enhance Notch signaling by interacting directly with NOTCH receptors. Consistent with a role in Notch signaling, NOTCH2NL ectopic expression delays differentiation of neuronal progenitors, while deletion accelerates differentiation into cortical neurons. Furthermore, NOTCH2NL genes provide the breakpoints in 1q21.1 distal deletion/duplication syndrome, where duplications are associated with macrocephaly and autism and deletions with microcephaly and schizophrenia. Thus, the emergence of human-specific NOTCH2NL genes may have contributed to the rapid evolution of the larger human neocortex, accompanied by loss of genomic stability at the 1q21.1 locus and resulting recurrent neurodevelopmental disorders