The label-free separation and culture of tumor cells in a microfluidic biochip.

Circulating tumor cells (CTCs) from liquid biopsy have shown a strong correlation to the clinical outcome of cancer patients. The enumeration and cytological analysis of CTCs have attracted increasing efforts for cancer disease management amid immunotherapy and personalized medicine. However, both enumeration and cytological analysis are challenging due to the rarity of CTCs and the lack of integrated solutions for the minimal risk of cell loss in the course of CTC procurement. We report a simple microfluidic chip permitting a one-stop solution for streamlining the on-chip cell separation, capture, immunofluorescence assay and/or in situ culture of isolated cells devoid of risky manual steps. Our results showed effective trapping of single cells, doublets and cell lumps isolated from blood in the same device. On-chip immunostaining revealed normal cell morphology and the characterization of cell expansion uncovered an altered cell growth curve with a reduced lag phase as compared to the conventional culture despite closely matching cell growth rates. The cells were viable and functional for as long as 11 days inside our chip and cell migration was also readily observed, with lumps showing greater aggressiveness than single cells. With these results, we expect promising applications of our one-stop solution for liquid biopsy via CTCs.

Olfactory deficit is associated with mitral cell dysfunction in the olfactory bulb of P301S tau transgenic mice.

Neurofibrillary tangles consisting of hyperphosphorylated tau (P-tau) are the neuropathological hallmark of Alzheimer's disease (AD), and olfaction disorder is an early symptom of AD. However, the link between P-tau aggregation and olfaction disorder remains unclear. In this study, the expression of P-tau in the olfactory bulb (OB), particularly in the mitral cell layer (MCL), external plexiform layer (EPL), and granule cell layer (GCL), of AD patients was found to be significantly higher than that in the OB of normal aging subjects, which suggested that these layers in the OB were susceptible to P-tau. The P301S tau transgenic mice (P301S mice) exhibit AD-like features, which can be characterized by olfactory dysfunction that precedes cognitive disorder. Importantly, the excessive P-tau expression in the OB of P301S mice, particularly in MCs, was associated with MC loss at 9 months of age, and decreased MC firing activities started to be observed at 2 months of age. Our results revealed that MCs might contribute to olfactory dysfunction in P301S mice. Furthermore, we described an aberrant dendro-dendritic synaptic structure between granule cells (GCs) and MCs and abnormal gamma oscillations in the EPL of the OB, and these findings indicated that P-tau might disrupt the regulation of MCs by GCs in P301S mice starting at 5 months of age. These data showed that the reduction in the MC firing frequency at 2 months of age might not be caused by GC suppression. Based on these findings, we speculated that MCs are a putative target for the treatment of P-tau-induced early olfactory dysfunction, and thus, an exploration of the specific causes and mechanisms of MC functional changes in P301S mice is crucial.

Impairment of Dendrodendritic Inhibition in the Olfactory Bulb of APP/PS1 Mice.

Olfactory dysfunction is an early event in Alzheimer's disease (AD). However, the mechanism underlying the AD-related changes in the olfactory bulb (OB) remains unknown. Granule cells (GCs) in the OB regulate the activity of mitral cells (MCs) through reciprocal dendrodendritic synapses, which is crucial for olfactory signal processing and odor discrimination. Nevertheless, the relationships between the morphological and functional changes of dendrodendritic synapses, particularly the local field potentials (LFPs) as a consequence of olfactory disorders in patients with AD have not been investigated. Here, we studied the morphological and functional changes induced by dendrodendritic inhibition in GCs onto MCs in the OB of amyloid precursor protein (APP)/PS1 mice and age-matched control mice during aging, particular, we focused on the effects of olfactory disorder in the dendrodendritic synaptic structures and the LFPs. We found that olfactory disorder was associated with increased amyloid-ß (Aß) deposits in the OB of APP/PS1 mice, and those mice also exhibited abnormal changes in the morphology of GCs and MCs, a decreased density of GC dendritic spines and impairments in the synaptic interface of dendrodendritic synapses between GCs and MCs. In addition, the aberrant enhancements in the γ oscillations and firing rates of MCs in the OB of APP/PS1 mice were recorded by multi-electrode arrays (MEAs). The local application of a GABAAR agonist nearly abolished the aberrant increase in γ oscillations in the external plexiform layer (EPL) at advanced stages of AD, whereas a GABAAR antagonist aggravated the γ oscillations. Based on our findings, we concluded that the altered morphologies of the synaptic structures of GCs, the dysfunction of reciprocal dendrodendritic synapses between MCs and GCs, and the abnormal γ oscillations in the EPL might contribute to olfactory dysfunction in AD.

Isolation of cells from whole blood using shear-induced diffusion.

Extraction of cells of interest directly from whole blood is in high demand, yet extraordinary challenging due to the complex hemodynamics and hemorheology of the sample. Herein, we describe a new microfluidic platform that exploits the intrinsic complex properties of blood for continuous size-selective focusing and separation of cells directly from unprocessed whole blood. The novel system only requires routinely accessible saline solution to form a sandwiched fluid configuration and to initiate a strong effect of shear-induced diffusion of cells, which is coupled with fluid inertia for effective separation. Separations of beads and cells from whole blood have been successfully demonstrated with high efficiency (89.8%) at throughput of 6.75 mL/hr (106-107 cells/s) of whole blood. Rapid isolation of circulating tumor cells (CTCs) from peripheral blood sample of hepatocarcinoma patients is also shown as a proof of principle.

Study of the union method of microelectrode array and AFM for the recording of electromechanical activities in living cardiomyocytes.

Electrophysiology and mechanics are two essential components in the functions of cardiomyocytes and skeletal muscle cells. The simultaneous recording of electrophysiological and mechanical activities is important for the understanding of mechanisms underlying cell functions. For example, on the one hand, mechanisms under cardiovascular drug effects will be investigated in a comprehensive way by the simultaneous recording of electrophysiological and mechanical activities. On the other hand, computational models of electromechanics provide a powerful tool for the research of cardiomyocytes. The electrical and mechanical activities are important in cardiomyocyte models. The simultaneous recording of electrophysiological and mechanical activities can provide much experimental data for the models. Therefore, an efficient method for the simultaneous recording of the electrical and mechanical data from cardiomyocytes is required for the improvement of cardiac modeling. However, as far as we know, most of the previous methods were not easy to be implemented in the electromechanical recording. For this reason, in this study, a union method of microelectrode array and atomic force microscope was proposed. With this method, the extracellular field potential and beating force of cardiomyocytes were recorded simultaneously with a low root-mean-square noise level of 11.67 µV and 60 pN. Drug tests were conducted to verify the feasibility of the experimental platform. The experimental results suggested the method would be useful for the cardiovascular drug screening and refinement of the computational cardiomyocyte models. It may be valuable for exploring the functional mechanisms of cardiomyocytes and skeletal muscle cells under physiological or pathological conditions.

Study of laser uncaging induced morphological alteration of rat cortical neurites using atomic force microscopy.

Activity-dependent structural remodeling is an important aspect of neuronal plasticity. In the previous researches, neuronal structure variations resulting from external interventions were detected by the imaging instruments such as the fluorescence microscopy, the scanning/transmission electron microscopy (SEM/TEM) and the laser confocal microscopy. In this article, a new platform which combined the photochemical stimulation with atomic force microscopy (AFM) was set up to detect the activity-dependent structural remodeling. In the experiments, the cortical neurites on the glass coverslips were stimulated by locally uncaged glutamate under the ultraviolet (UV) laser pulses, and a calcium-related structural collapse of neurites (about 250 nm height decrease) was observed by an AFM. This was the first attempt to combine the laser uncaging with AFM in living cell researches. With the advantages of highly localized stimulation (<5 µm), super resolution imaging (<3.8 nm), and convenient platform building, this system was suitable for the quantitative observation of the neuron mechanical property variations and morphological alterations modified by neural activities under different photochemical stimulations, which would be helpful for studying physiological and pathological mechanisms of structural and functional changes induced by the biomolecule acting.

[Research progress of external tufted cells in olfactory glomerulus].

External tufted (ET) cells are the major excitatory elements coordinating the activities of glomerulars and mediating the input from the olfactory neurons to mitral cells. The ET cells participate in inter-and intra-glomerular microcircuits in the olfactory bulb, link the isofunctional odor columns within the same olfactory bulb, and play an important role in olfactory information processing. This paper reviews the research progress of the anatomy and physiological properties and electrophysiological modeling of ET cells, elaborate the problems and defects in the field. And then it further gives some proposals for the future research of electrophysiological properties, development of olfactory information coding and performance of modeling of ET cells.

The fabrication of an olfactory receptor neuron chip based on planar multi-electrode array and its odor-response analysis.

A novel olfactory neurochip based on olfactory receptor neurons (ORNs) cultivated on the surface of the 60-channel planar multi-electrode array (MEA) devices was developed in this study. In order to investigate the odor-response characteristics of ORNs, two types of odorants at different concentrations were quantitatively pumped into the neurochip by a customized gas intake system, and the extracellular electrical activities of multiple ORNs were simultaneously recorded in vitro. Accordingly, the odor-response features of ORNs such as firing amplitude, firing threshold, firing rate as well as firing channels were analyzed qualitatively and quantitatively in terms of ORN spike trains. Especially, after introducing the classification algorithm based on the spike threshold, the odor-response maps from the multiple sites could be used to identify dl-limonene (LIM) and isoamyle acetate (ISO) odorants. These preliminary studies indicate that the ORN-based biosensor developed here has a potential capability of distinguishing different odorants as a true bionic electronic nose.