We present a novel method, implemented in the form of a microfluidic device, for arraying and analyzing large populations of single cells. The device contains a large array of electroactive ...microwells where manipulation and analysis of large population of cells are carried out. On the device, single cells can be actively trapped in the microwells by dielectrophoresis (DEP) and then lysed by electroporation (EP) for subsequent analysis of the confined cell lysates. The DEP force in the selected dimensions of the microwells could achieve efficient trapping in nearly all the microwells (95%) in less than three minutes. Moreover, the positions of the cells in the microwells are maintained even when unstable flow of liquid is applied. This makes it possible to exchange the DEP buffer to a solution that will be subsequently used for stimulating or analyzing the trapped cells. After closing the microwells, EP is conducted to lyse the trapped cells by applying short electric pulses. Tight enclosure is critical to prevent dilution, diffusion and cross contamination of the cell lysates. We demonstrated the feasibility of our approach with an enzymatic assay measuring the intracellular‐galactosidase activity. The use of this method should greatly help analysis of large populations of cells at the single‐cell level. Furthermore, the method offers rapidity in the trapping and analysis of multiple cell types in physiological conditions that will be important to ensure the relevance of single cell analyses.
Electroactive microwells attract a single cell per well by inducing dielectrophoresis, and lyse trapped cells with electroporation for subsequent analysis of the intracellular constituents. An array of the electroactive microwells is used for high‐throughput and parallelized read‐outs of individual intracellular‐β‐galactosidase levels in a large population of cells.
Testing of drug effects and cytotoxicity by using cultured cells has been widely performed as an alternative to animal testing. However, the estimation of pharmacokinetics by conventional cell-based ...assay methods is difficult because of the inability to evaluate multiorgan effects. An important challenge in the field is to mimic the organ-to-organ network in the human body by using a microfluidic network connecting small-scale tissues based on recently emerging MicroTAS (Micro Total Analysis Systems) technology for prediction of pharmacokinetics. Here, we describe an on-chip small intestine-liver coupled model for pharmacokinetic studies. To construct an in vitro pharmacokinetic model that appropriately models in vivo conditions, physiological parameters such as the structure of internal circulation, volume ratios of each organ, and blood flow ratio of the portal vein to the hepatic artery were mimicked using microfluidic networks. To demonstrate interactions between organs in vitro in pharmacokinetic studies, Caco-2, HepG2, and A549 cell cultures were used as organ models of the small intestine, liver, and lung, respectively, and connected to each other through a microporous membrane and microchannels to prepare a simple model of a physiological organ-to-organ network. The on-chip organ model assay using three types of substrate-epirubicine (EPI), irinotecan (CPT-11), and cyclophosphamide (CPA)-were conducted to model the effects of orally administered or biologically active anticancer drugs. The result suggested that the device can replicate physiological phenomena such as activity of the anticancer drugs on the target cells. This microfluidic device can thus be used as an in vitro organ model to predict the pharmacokinetics of drugs in the human body and may thus provide not only an alternative to animal testing but also a method of obtaining parameters for in silico models of physiologically based pharmacokinetics.
In contrast to cell cultures, particularly to cell lines, tissues or organs removed from the body cannot be maintained for long in any culture conditions. Although it is apparent that in vivo ...regional homeostasis is facilitated by the microvascular system, mimicking such a system ex vivo is difficult and has not been proved effective. Using the culture system of mouse spermatogenesis, we addressed this issue and devised a simple microfluidic device in which a porous membrane separates a tissue from the flowing medium, conceptually imitating the in vivo relationship between the microvascular flow and surrounding tissue. Testis tissues cultured in this device successfully maintained spermatogenesis for 6 months. The produced sperm were functional to generate healthy offspring with micro-insemination. In addition, the tissue kept producing testosterone and responded to stimulation by luteinizing hormone. These data suggest that the microfluidic device successfully created in vivo-like conditions, in which testis tissue maintained its physiologic functions and homeostasis. The present model of the device, therefore, would provide a valuable foundation of future improvement of culture conditions for various tissues and organs, and revolutionize the organ culture method as a whole.
Three-dimensional aggregation and organ culture methods are critical for recreating in vivo cellular phenomena outside the body. Previously, we used the conventional gas liquid interphase organ ...culture method to induce complete mouse spermatogenesis. After incorporating microfluidic systems, we achieved a significant increase in efficiency and duration of spermatogenesis. One of the major drawbacks preventing the popularization of microfluidics, however, is the use of a power-pump to generate medium flow. In this study, we produced a pumpless microfluidic device using hydrostatic pressure and a resistance circuit to facilitate slow, longer lasting medium flow. During three months of culture, results in induction and maintenance of spermatogenesis showed no difference between pumpless and pump-driven devices. Correspondingly, the spermatogonial population was favorably maintained in the pumpless device compared to the conventional method. These results show the advantage of using microfluidic systems for organ culture experiments. Our pumpless device could be applied to a variety of other tissues and organs, and may revolutionize organ culture methods as a whole.
Genetic analysis, rather than simply counting the number of circulating tumor cells (CTCs), which are rare cancer cells in peripheral blood, has great potential for non-invasive biopsy or "liquid ...biopsy." However, a practical problem in conventional enrichment of CTCs is that the isolated target cells are mixed with numerous residual leukocytes, and are suspended in a large volume. Hence, further isolation (i.e., cytokeratin (CK)-positive cell picking) or DNA purification is required for downstream genetic analysis after isolation. Here, we propose a novel cancer marker-free method of CTC enrichment by size-based Filtration and Immunomagnetic Negative selection followed by Dielectrophoretic concentration (CTC-FIND) for direct detection of genetic mutations in rare cancer cells suspended in whole blood. A combination of two independent isolation methods based on physical (filtration) and biochemical properties (immunomagnetic negative selection) in CTC-FIND allowed highly efficient cancer marker-free purification (5.1-log depletion of leukocytes). The isolated cells were trapped and concentrated using a microfluidic step-channel device using dielectrophoresis for discrimination and downstream genetic analysis. The feasibility of cancer marker-free enrichment by CTC-FIND was successfully demonstrated by directly detecting mutations in various cancer cells with a very high sensitivity of 1 cell per mL, including EpCAM and CK-negative cells, which were used to spike 8 mL of whole blood. Thus, CTC-FIND can be used with liquid biopsy to detect genetic mutations in wide-ranging CTC subsets, independent of cancer cell-specific marker expression.
Graphical abstract Highlights ► Nucleic acids can be used to build reaction modules modeled after in vivo or in silico computers. ► In vitro circuits built from these modules perform one-shot ...calculations or show complex dynamics. ► Applications at the interface with biology, or for molecular-scale robotics, are burgeoning.
In this study, we propose a microfluidic cell culture device mimicking the microscopic structure in liver tissue called hepatic cords. The cell culture area of the device was designed to align ...hepatocytes in two lines in a similar way to hepatic cords. Thanks to the structural design together with a cell seeding procedure, rat primary hepatocytes were successfully aligned in two lines and cultured under perfusion condition. It is shown that aligned hepatocytes gradually self-organize and form bile canaliculi along the hepatic cord-like structure. The present technique to culture hepatocytes with functional bile canaliculi could be used as an alternative to animal testing in the field of drug discovery and toxicological studies, and also be beneficial to tissue engineering applications.
Mouse spermatogenesis, from spermatogonial stem cell proliferation to sperm formation, can be reproduced in vitro by culturing testis tissue masses of neonatal mice. However, it remains to be ...determined whether this method is also applicable when testis tissues are further divided into tiny fragments, such as segments of the seminiferous tubule (ST), a minimal anatomical unit for spermatogenesis. In this study, we investigated this issue using the testis of an Acrosin-GFP/Histone H3.3-mCherry (Acr/H3) double-transgenic mouse and monitored the expression of GFP and mCherry as indicators of spermatogenic progression. Initially, we noticed that the cut and isolated stretches of ST shrunk rapidly and conglomerated. We therefore maintained the isolation of STs in two ways: segmental isolation without truncation or embedding in soft agarose. In both cases, GFP expression was observed by fluorescence microscopy. By whole-mount immunochemical staining, meiotic spermatocytes and round and elongating spermatids were identified as Sycp3-, crescent-form GFP-, and mCherry-positive cells, respectively. Although the efficiency was significantly lower than that with tissue mass culture, we clearly showed that spermatogenesis can be induced up to the elongating spermatid stage even when the STs were cut into short segments and cultured in isolation. In addition, we demonstrated that lowered oxygen tension was favorable for spermatogenesis both for meiotic progression and for producing elongating spermatids in isolated STs. Culturing isolated STs rather than tissue masses is advantageous for explicitly assessing the various environmental parameters that influence the progression of spermatogenesis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
During development, axons spontaneously assemble into a fascicle to form nerves and tracts in the nervous system as they extend within a spatially constrained path. However, understanding of the ...axonal fascicle has been hampered by lack of an in vitro model system. Here, we report generation of a nerve organoid composed of a robust fascicle of axons extended from a spheroid of human stem cell-derived motor neurons within our custom-designed microdevice. The device is equipped with a narrow channel providing a microenvironment that facilitates the growing axons to spontaneously assemble into a unidirectional fascicle. The fascicle was specifically made with axons. We found that it was electrically active and elastic and could serve as a model to evaluate degeneration of axons in vitro. This nerve organoid model should facilitate future studies on the development of the axonal fascicle and drug screening for diseases affecting axon fascicles.
•Axons spontaneously assembled into a unidirectional fascicle within a microchannel•The axon fascicles were electrically active and elastic•The axon fascicle can model degeneration of axons in vitro
In this article, Ikeuchi and colleagues show that axons of stem cell-derived motor neurons spontaneously assembled into a unidirectional fascicle within a microchannel. The axon fascicles were electrically active and elastic and can model degeneration of axons in vitro. This model could facilitate studies on the development of the axon fascicles and drug screening for diseases affecting axon fascicles.
Podocytes, localized in the glomerulus, are a prognostic factor of proteinuria in kidney disease and are exposed to distinct physiological stimuli from basal to apical filtration flow. Research ...studies on drug discovery and disease modeling for glomerulopathy have developed a glomerulus-on-a-chip and studied podocyte mechanobiology to realize alternative methods to animal experiments. However, the effect of filtration stimulus on podocytes has remained unclear. Herein, we report the successful development of a user-friendly filtration culture device and system that can precisely control the filtration flow using air pressure control by incorporating a commercially available culture insert. It allows mouse podocytes to be cultured under filtration conditions for three days with a guarantee of maintaining the integrity of the podocyte layer. Using our system, this study demonstrated that podocyte damage caused by hyperfiltration resulting from glomerular hypertension, a common pathophysiology of many glomerulopathies, was successfully recapitulated and that filtration stimulus promotes the maturation of podocytes in terms of their morphology and gene expression. Furthermore, we demonstrated that filtration stimulus induced different drug responsiveness in podocytes than those seen under static conditions, and that the difference in drug responsiveness was dependent on the pharmacological mechanism. Overall, this study has revealed differentiating and pharmacodynamic properties of filtration stimulus and brings new insights into the research field of podocyte mechanobiology towards the realization of glomerulus-on-a-chip.
The development of a filtration flow device system with precisely controlled pressure revealed the mechanobiology of filtration flow to podocytes regarding morphological and gene expression maturation and increased sensitivity to toxic drugs.
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