In this paper, we construct a microfluidic device with an embedded pressure sensor to study the in-plane direction elasticity of adenocarcinomic human alveolar epithelial (A549) cells layer with different oncogene, multiple copies in T-cell malignancy (MCT-1), expression levels [1].  The pressure sensor is constructed based on electrofluidic circuits, ionic liquid-filled microfluidic channel networks.  The device consists of three polydimethylsiloxane (PDMS) layers: a top cell culture chamber layer, a middle sensing membrane layer and a bottom electrofluidic circuit layer as shown in Figure 1(a). On the top layer, a cell culture chamber with a single inlet and a single outlet is designed to culture cells for the measurement.  On the bottom ionic liquid-filled circuit layer, four identical electrofluidic resistors [2] designed and arranged as a Wheatstone bridge circuit as shown in Figure 1(b). An elastic sensing membrane is sandwiched between the top and bottom layers.  When the sensing membrane is deformed by pressure application, the geometries of the electofluidic channel will be changed, and the characteristic of the electrofluidic circuit will also be changed accordingly. The change will further vary the output voltage signal from the circuit. When the cells are seeded on the top of the sensing membrane, the cell-adhered membrane can be modeled as a two-layer composite plate. To quantitatively estimate the in-plane elasticity of a layer of cells, we derive a theoretical model based on first order shear deformation theory of plate (FSDT) [3-4] and basic circuit theories to estimate the cell elasticity from the sensor sensitivity variation. For comparison, we use the atomic force microscope (AFM) to measure the out-of-plane elasticity and thickness of the A549 cell layers. The average measured thickness of A549 cells is 1.11μm. With the measured pressure sensor output signals and the sensing membrane geometries and mechanical properties, we can calculate the relationship between the Young’s modulus of the cells layer and the sensitivity ratio.   The ratio is obtained from the same device with and without the cells cultured in it (Figure 2). In the experiments, A549-control cells (A549-C) and A549 cells with MCT-1 oncogene overexpression (A549-M) [5] are used to investigate their in-plane elasticities. Figure 3 shows the bright field phase images of the A549 cells cultured in the microfluidic devices during the experiment. Figure 4 (a) and Figure 4 (b) show the typical time-lapsed sensitivity variation of the devices cultured A549-C and A549-M cells, respectively.  According to the Figure 2, we can estimate the in-plane elasticity of A549-C and A540-M cells layers.  Figure 5 shows comparison of the average in-plane elasticities of the A549-C and A549-M cells measured using the developed microfluidic devices. The results show that the average in-plane elasticities of A549-C and A549-M are 10.51 MPa and 20.87 MPa (n=3), respectively. The result demonstrates that the device can successfully measure the in-plane elasticity of the cells, and the elasticity increases when MCT-1 oncogene overexpressed in the A549 cells. With the demonstrated capability, the developed device shows its great potential for study of cell physical properties with different gene expression.

Tumor metastasis causes the death in most cancer patients. During metastasis, cell migrated from primary tumor was the very first step and study the tumor cell migration and interaction with somatic cells is therefore of great important. Development of in vitro technology that can quantitatively gain migration and cell-cell interaction data is there high desirable. Conventional methods for study tumor migration and cell-cell interaction are plagued by inaccessible to get quantitative data or complication of microfabrication process which brings into additional efforts rather than study the biological question itself. Herein, we developed a 3D printed “LEGO”-like device combined with a quantitative data gaining process which can be used for investigation into tumor cell migration process dynamically at single cell resolution and tumor-stromal interactions with high flexibility.

Circulating tumor cells (CTCs) are cancer cells shed from primary tumor and circulating in the peripheral blood. CTCs initiate secondary tumor colonies and result in more than 90% of cancer death. Isolation and enumeration of CTCs have exhibited promising clinical applications in cancer early diagnosis, prognosis and personalized therapy. However, owing to the extremely low rarity (as low as one CTC in one billion blood cells), CTCs isolation remains a great technical challenge. Current CTCs isolation approaches are based either on their biological properties, including immunoassay, magnetic-activated cell sorting, fluorescence-activated cell sorting, or physical properties, including filtering, inertial microfluidics, deterministic lateral displacement and etc. In general, biology-based techniques are characterized as high purity but low throughput; while physical property based techniques have advantages of low cost and high throughput. In this work, we reported the fabrication of a multilayer crossflow device and demonstrated the capability of isolating CTCs from whole blood based on lateral-flow filtration. The device was consisted of a filter membrane sandwiched between two microfluidic channel layers and fabricated with standard PDMS soft lithography and multilayer bonding technique. A 77.3% filtration efficiency with 4.5um polystyrene beads and a 97% cancer cells recovery rate have been achieved for a single-pass filtration. To further enhance the enrichment ratio(concentration of CTCs prior to filtration over that post-filtration) of CTCs over blood cells, a recirculating filtration system was built by combing the crossflow device with pressure pump and electromagnetic valves. In this system, 1 ml blood sample was treated for 10 times within 40 minutes, with an enrichment ratio of more than 10⁴.

        This paper reports a new application of microfluidic devices in optogenetics and the study of learning behavior of model organisam. Due to the fully known genomic information and neuronal connections, Caenorhabditis elegans have been chosen as objects to study the preference establishment and their learning ability. The optogenetics-microfluidics manipulation platform provides a novel measurement method to quantify C.elegans behaviors and largely improve the experiment efficiency[1][2]. 

        In our research, two channelrhodopsins, Chrimson and CoChR, are expressed in the pair of olfactory receptor neurons AWA and AWB, as well as the Ca2+ indicator, GEM-GECO protein. Modified worms were endued the artificial phenotype of heading for the blue light ray but avoiding the red light. Synchronic worms are selected and injected into the microfluidics devices. After operant conditioning and reinforcement training using laser excitation at specific wavelengths, their behaviors of moving forward or backward could be observed when worms semi-fixed in PDMS chambers. Corresponded neuron activities are indicated by the fluorescence intensity of GEM-GECO. Their responds to the odor of diacetyl and nonanone are also recorded. Data is collected to analyze their behavior deviation and adaptability under different conditioned stimulus.

       The reprogramming chemotaxis responses of AWA, AWB were reported in 1997 [1]. It was also the first discovery of the determinant of olfactory preference. Based on the new optogenetics methods[3] and microfluidics devices[4], we study the probability to deviate their natural preference. Compared with conventional methods basically relied on chemical stimulation and behavioral statics[5][6], the optical activation of neurons is more fast and accurate. An overview of the responding mechanism in our neuron cell is shown in picture 1, and the schematic view of the microfluidics devices is in Figure 2, 3 and 4. The picture showing the position of olfactory receptor neurons is 5, and the excitation spectra of proteins in this research is shown in picture 6.

REFERENCES: 

[1] Emily R. Troemel, Bruce E. Kimmel and Cornelia I. Bargmann, "Reprogramming chemotaxis responses: sensory neurons define olfactory preferences in C.elegans," J. Cell. 1997, Vol. 91, 161–169.

[2] Steven J. Husson, Alexander Gottschalk and Andrew M. Leifer, "Optogenetic manipulation of neural activity in C. elegans: From synapse to circuits and behavior," J. Cell. 2013. Vol. 105, 235-250.

[3] Nathan C Klapoetke, Yasunobu Murata et al, "Independent optical excitation of distinct neural populations," J. Nature Methods. 2014. Vol.11, No.3.

[4] Adriana San-Miguel and Hang Lu, "Microfluidics as a tool for C.elegans’ research." Wormbook. 2013.

[5] Ye, Hua-Yue, Bo-Ping Ye, and Da-Yong Wang. "Learning and learning choice in the nematode Caenorhabditis elegans." Neuroscience bulletin 22.6 (2006): 355-360.

[6] Ardiel, Evan L., and Catharine H. Rankin. "An elegant mind: learning and memory in Caenorhabditis elegans." Learning & Memory 17.4 (2010): 191-201.

The growing emphasis on personalized medicine significantly increases the need to analyze key molecular markers. In comparison to tissue biopsies, circulating biomarkers (liquid biopsies) can be conveniently and repeatedly obtained from biofluids with minimal complications. In particular, exosomes have recently emerged as a promising circulating biomarker. Exosomes are nanometer-sized membrane vesicles actively shed off by cells and possess unique advantages: they abound in biofluids, readily cross the blood brain barrier and harbor diverse molecular contents. A sensor platform capable of sensitive and rapid detection of exosomes would thus be an invaluable tool in translating their clinical potential. In this talk, I will describe various systems that we have developed for quantitative analyses of circulating cancer exosomes. By enabling rapid, sensitive and cost-effective detection of circulating biomarkers, these platforms could significantly expand the reach of preclinical and clinical research, in informing therapy selection, rationally directing trials, and improving sequential monitoring to achieve better clinical outcomes.

Drug testing applications for efficacy and safety testing in vitro with animal and human cells in various tissue-mimic constructs is gaining momentum in the forms of organ-on-chips, micro patterned populations, organoids etc. Most of these constructs face mass transfer and drug access issues that are well-addressed by micro-systems engineering. However, such systems, though allowing precision controls of microenvironment and the associated benefits such as the miniature sample sizes, there are significant challenges on measurements, scalable techniques, consistency, and robustness, and such issues are becoming more severe when complexity increases in multiple cell-type co-culture configurations that are becoming popular. Here we will illustrate a few configurations of increasing complexity of micro-system engineered drug testing platforms to illustrate these challenges and thus stimulate further innovations to address these issues for practical applications in various forms of in vitro drug testing in academia and industry. The examples will range from micropatterned human embryonic stem cell-based developmental toxicity testing platform in testing 35 compounds, to a bile canaliculi-based contraction assay for testing drug against cholestasis, to microfluidics co-cultured system for testing nutraceuticals against cardiovascular diseases, among others.

Blood vessel construction is a very significant and difficult research aspect in tissue engineering field. In this paper, we present a method for producing vascular networks which assembled multi-layer of cells onto silicified tubular chip taking from mouse or other animals. For mimicking the complex tissue structure in vitro, there are already some outstanding works have been reported. For example, Xinyu Jiang’s group has reported a method for stepwise formation of 2D multicellular structures through the biotin-streptavidin (SA) interaction and further construction of controlled, 3D, multilayered, tissue-like structures using the stress-induced rolling membrane (SIRM) technique [1], and Zev J Gartner's group described a DNA-programmed assembly of cells method to programmed synthesize three-dimensional tissues[2].

Here, in order to form vascular networks in vitro, we first obtain a multi-branched tubular tissue from the body as scaffold, and converting it into silica materials through silica deposition after acellular treating the tissue by detergents. The click chemistry and DNA assembly method were used to construct cell layer on the surface of the silica-tubular, and by repeating the DNA assembly procedure we have successfully assembled multilayer cells. Furthermore, we implanted them into collagen to produce in vitro vascular network induced by vascular endothelial growth factor, finally， a complex, multilayer cell coated and branched vascular network has been constructed in collagen.

A schematic view of the experiment is shown in Figure 1.The resulting silicified vascular shown in Figure 2 holds a high hardness for operation and topological structures which benefits the cell growth and proliferation. In summary, a vascular network has been constructed by our method and we believe other tissues can also be assembled in this way.

In this paper, we propose a microfluidic device synthesizing Au nanorods based on photochemical reduction method. It is expected that we can achieve high uniformed Au nanorods in size and shape through the microfluidic device based synthesis.

 In the microfluidic device, we applied passive mixers to achieve more effective chemical reactions for Au nanorods synthesis. We also demonstrate effective passive mixer designed by the finite element method (FEM) simulation study.

This study integrated the microfluidic system and ODEP technology for the isolation of CTC clusters from the background leukocytes. The working principle is based on the size difference between the CTC clusters and leukocytes, and thus different magnitude of ODEP force acting on them. ODEP mainly use a controllable light pattern, acting as a virtual electrode, to generate a non-uniform electric field that is in turn utilized to manipulate the electrically-polarized cells. The utilization of ODEP-based mechanism for CTCs isolation has been successfully demonstrated in our previous study [1].

Since 1970, a series of clinical studies have shown that single CTCs may not be the main cause of cancer metastasis, but two or more aggregated CTCs [2, 3]. In order to isolate CTC clusters for back-end analysis. For the biological-based methods (e.g. ^HBCTC-Chip [4], or CTC-iChip [5]), although CTC clusters can be specifically separated by antibody-based schemes, but the surface-area-to-volume ratio of CTC clusters is relatively low which might affect the binding efficiency of CTC clusters and antibody. Alternatively, some studies proposed physical-based methods to separate CTC clusters (e.g. ISET [6], FMSA [7], Cluster-chip [8]). Although these methods have been demonstrated to effectively isolate the CTC clusters from the background cells mainly based on their size difference, the influence of shear stress on physical size of CTC cluster, or the viability of the cells isolated is still a problem.

To address this issue, The key advantages of ODEP mechanism for cell isolation including: (1) no need of complex microfabrication process for constructing microfilter structures, and (2) the reduction of shear stress acting on the cells manipulated.

However, the feasibility of using ODEP-based mechanism for the isolation of CTC cluster (i.e. CTC cell aggregates) has not yet been explored. To test its feasibility, a T-shaped microfluidic chip was designed (Fig.1). A virtual microfilter consisting of multiple light patterns was designed at the CTC clusters isolation zone (Fig.1). By continuously moving and rotating the light patterns in the microfilter, the larger CTC clusters can be separated from the background leukocytes, and also transported to the side microchannel (Fig.2). In this work, the optimum ODEP operating conditions (e.g. moving velocity of light pattern) was explored. Results revealed that moving velocity of light pattern that can manipulate the CTC clusters (containing 2-13 cells) was significantly higher than the background of leukocytes (Fig.3). Based on this, the moving velocity of light pattern was set at 100 μm/sec (Fig.3). At a given sample flow rate of 0.5 μl/min, moreover, we found that the rotation speed of light patterns at 14 RPM could significantly increase the purity of CTC clusters isolation (Fig.4). Based on the set operating conditions, the recovery and purity of the isolated CTC clusters were experimentally evaluated to be 70.1 ± 7.1% and 60.8 ± 2.7%, respectively (Fig.4). As a whole, we have established a high purity CTC clusters isolation method that is easy to operate, and is possible to avoid the problem caused by the shear stress acting on the cells or particles.

Point of care testing (POCT) devices are expected for daily healthcare, bedside monitoring and so on. They also expected for agricultural fields such as health monitoring of livestock. For example monitoring of progesterone concentration in blood or milk of cow is important to detect estrus cycle, pregnancy, matstitis, and so on [1]. The detections contribute to improve the production efficiency of cattle. In this field, immunosensor, which is inexpensive, available in the fields, easy to use even by farmers and quantitative capability, is required. Therefore, we developed POCT device by combining immunochromatography and electrochemical method, and measured progesterone with the POCT device.

Figure 1 shows photograph (Fig. 1a) and schematic of cross-sectional view (Fig. 1b) of developed POCT device [2]. The device consists of nitrocellulose membrane, absorbent pad, electrochemical detector, and two polymethylmethaclylate (PMMA) plates. Nitrocellulose membrane is attached to the electrochemical detector and they are sandwiched with two PMMA plates. Figure 1c shows schematic of electrochemical detector. Gold working electrode (WE), Ag/AgCl reference electrode (RE), and gold counter electrode (CE) are displaced in order from upstream with 0.5 mm intervals. WE and RE has 1 mm width and CE has 2 mm width.

Figure 2 shows experimental procedure to measure progesterone. Progesterone was measured by competitive immunoassay. At first, progesterone and biotin labeled progesterone were injected to nitrocellulose membrane. Unlabeled progesterone competed with labeled progesterone to bind to antibody. Unbound progesterone were washed away by next washing step. After that, streptavidin labeled alkaline phosphatase (ALP) was injected. The streptavidin bound to biotin and formed conjugate. Unbound ALP was also washed away by second washing step. Finally, p-aminophenyl phosphate (pAPP) was injected and reacted with ALP and produced p-amino phenol (pAP). The amount of pAP was measured by electrochemical detector. Chronoamperometry with applied potential was 0.2 V vs Ag/AgCl was started when the pAPP was injected to the membrane. Figure 3a shows amperometric signal of each concentration of progesterone. Each signal shows peak signal. It is considered that the peak signal obtained when pAP reached to the detector. The height of peak oxidation current was correlated with concentraton of progesterone (Fig. 3b). This results suggests that our device can measure the concentration of progesterone.

This work was partly supported by the Council for Science, Technology and Innovation (CSTI), Cross-ministerial Strategic Innovation Promotion Program (SIP), “Technologies for creating next-generation agriculture, forestry and fisheries” (funding agency: Bio-oriented Technology Research Advancement Institution, NARO) and by the Japan Society for the Promotion of Science KAKENHI Grant number 16K06036.