Single-Cell Chemical Lysis on Microfluidic Chips with Arrays of Microwells

Many conventional biochemical assays are performed using populations of cells to determine their quantitative biomolecular profiles. However, population averages do not reflect actual physiological processes in individual cells, which occur either on short time scales or nonsynchronously. Therefore, accurate analysis at the single-cell level has become a highly attractive tool for investigating cellular content. Microfluidic chips with arrays of microwells were developed for single-cell chemical lysis in the present study. The cellular occupancy in 30-μm-diameter microwells (91.45%) was higher than that in 20-μm-diameter microwells (83.19%) at an injection flow rate of 2.8 μL/min. However, most of the occupied 20-μm-diameter microwells contained individual cells. The results of chemical lysis experiments at the single-cell level indicate that cell membranes were gradually lysed as the lysis buffer was injected; they were fully lysed after 12 s. Single-cell chemical lysis was demonstrated in the proposed microfluidic chip, which is suitable for high-throughput cell lysis

Advanced 3D Printing to Fabricate Microfluidic Devices for Cancer and Stem Cells Co-culture Study in Space

Two different type of microfluidic devices were designed, fabricated and tested to capture the microspheres. The passive device seems to be more reliable because of no possibility of damage, whereas the thin film in active device got ruptured when too much pressure was applied to the valve control layer. The passive design was able to capture microspheres of different sizes. Majority of microspheres captured were between 150-175 microns. The capture efficiency for this device was slightly lower than expected at 26%. This was found to be due to the long channel length which leads to pressure drop towards the end of the channel. In addition, capturing of microspheres causes high resistance to flow towards the end of channel. To the best of my knowledge, this is a first kind of device to capture microspheres at this size range of 125-215 microns. The proof of concept for capturing large particle size >100um and broad size distribution has been demonstrated. The device will be further improved by optimizing the dimensions

Advanced 3D Printing to Fabricate Microfluidic Devices for Cancer and Stem Cells Co-culture Study in Space

Two different type of microfluidic devices were designed, fabricated and tested to capture the microspheres. The passive device seems to be more reliable because of no possibility of damage, whereas the thin film in active device got ruptured when too much pressure was applied to the valve control layer. The passive design was able to capture microspheres of different sizes. Majority of microspheres captured were between 150-175 microns. The capture efficiency for this device was slightly lower than expected at 26%. This was found to be due to the long channel length which leads to pressure drop towards the end of the channel. In addition, capturing of microspheres causes high resistance to flow towards the end of channel. To the best of my knowledge, this is a first kind of device to capture microspheres at this size range of 125-215 microns. The proof of concept for capturing large particle size >100um and broad size distribution has been demonstrated. The device will be further improved by optimizing the dimensions

Microtechnologies for Cell Microenvironment Control and Monitoring

A great breadth of questions remains in cellular biology. Some questions cannot be answered using traditional analytical techniques and so demand the development of new tools for research. In the near future, the development of highly integrated microfluidic analytical platforms will enable the acquisition of unknown biological data. These microfluidic systems must allow cell culture under controlled microenvironment and high throughput analysis. For this purpose, the integration of a variable number of newly developed micro- and nano-technologies, which enable control of topography and surface chemistry, soluble factors, mechanical forces and cell-cell contacts, as well as technology for monitoring cell phenotype and genotype with high spatial and temporal resolution will be necessary. These multifunctional devices must be accompanied by appropriate data analysis and management of the expected large datasets generated. The knowledge gained with these platforms has the potential to improve predictive models of the behavior of cells, impacting directly in better therapies for disease treatment. In this review, we give an overview of the microtechnology toolbox available for the design of high throughput microfluidic platforms for cell analysis. We discuss current microtechnologies for cell microenvironment control, different methodologies to create large arrays of cellular systems and finally techniques for monitoring cells in microfluidic devices.E.A.-H. acknowledges funding from the Basque Government, Department of Education, for predoctoral fellowship 2016. M.G.-H. acknowledges funding from the University of the Basque Country UPV/EHU, PIF16/204 predoctoral fellowship "call for recruitment of research personnel in training". J.E.-E. acknowledges funding from the University of the Basque Country UPV/EHU, postdoctoral fellowship ESPPOC 16/65 "Call for recruitment and specialization of Doctor Researchers 2016". M.M.D.P. and L.B.-D., acknowledge funding support from University of the Basque Country UPV/EHU, UFI11/32, and from Gobierno Vasco under Grupos Consolidados with Grant No. IT998-16. F.B.-L. acknowledges funding support from the Ramon y Cajal Programme (Ministerio de Economia y Competitividad), Spain. F.B.-L. and L.B.-D. acknowledge funding support from the European Union's Seventh Framework Programme (FP7) for Research, Technological Development and Demonstration under Grant agreement No. 604241 as well as Gobierno Vasco, Dpto. Industria, Innovacion, Comercio y Turismo under ELKARTEK 2015 with Grant No. KK-2015/0000088

Cell Microarray Technologies for High-Throughput Cell-Based Biosensors

Due to the recent demand for high-throughput cellular assays, a lot of efforts have been made on miniaturization of cell-based biosensors by preparing cell microarrays. Various microfabrication technologies have been used to generate cell microarrays, where cells of different phenotypes are immobilized either on a flat substrate (positional array) or on particles (solution or suspension array) to achieve multiplexed and high-throughput cell-based biosensing. After introducing the fabrication methods for preparation of the positional and suspension cell microarrays, this review discusses the applications of the cell microarray including toxicology, drug discovery and detection of toxic agents.ope

Quantification of microRNA expression in single cells using microfluidics

Chronic obstructive pulmonary disease (COPD) is a lung condition characterised by progressive airflow limitation in part due to narrowing and fibrosis of small airways. COPD is associated with cellular senescence which is driven by stressors such as oxidative stress. MicroRNA-21 (miR-21) and microRNA-34a (miR-34a) are upregulated in COPD, however their regulatory role in COPD pathogenesis or in response to oxidative stress remains unclear. To better understand the role of miR-21 and miR-34a in COPD, a microfluidic platform was developed to quantify miR-21 and miR-34a molecules in single cells. Sandwich hybridisation assay was optimised and integrated into microfluidic chambers for single cell miRNA detection. The sensitivity was demonstrated by quantifying levels of miRNA in nasal cells and fluid. Levels of miR-21 were varied in nasal cells and fluid within and between individuals. Levels of miR-21 and miR-34a were increased in small airway epithelial cells (SAEC) and fibroblasts (SAF) from COPD subjects compared to non-smokers, and were varied within and between subjects. MiR-21 and miR-34a were detected simultaneously from the same cell using a multiplex assay which showed a positive correlation between miR-21 and miR-34a expressed in SAEC and SAF. Baseline gene expression of miR-21 and miR-34a targets differed in SAEC and SAF. Altered levels of miR-21 and miR-34a influenced their targets mRNA and protein levels, however the effect was different in COPD cells compared to healthy cells. MiR-21 and miR-34a levels were elevated in response to oxidative stress, while their target gene expression was reduced and senescent markers were increased. This study demonstrated that a microfluidic platform can be developed to quantify single miRNA molecules in single cells to determine cell-to-cell variation of miRNAs within cell populations. MiR-21 and miR-34a are crucial regulators in COPD and may have a protective role in response to oxidative stress, however further investigation is required.Open Acces

A microwell array coated with dopaminergic cell adhesive film for single cell analysis in drug discovery

Drug discovery requires prompt decision-making to identify which new chemical entities constitute viable new drug candidates and have a high likelihood of market success. Conventional in vitro cell-based screens sometimes provide misleading data that may not represent in vivo responses. We developed an array of microcellular platforms to provide a uniform environment for single-cell suspension, mimic in vivo functions, and demonstrate the biological effects of a drug on the chemistry of a single cell at the molecular level. Dopaminergic mesoporous inorganic-organic hybrid resin (HR4-DOPA) was used to coat the wells of 200-microwell plates fabricated for these experiments. The biocompatibility of HR4-DOPA was demonstrated by cell adhesion and viability assays. HeLa cell adhesion to the HR4-DOPA film was comparable to the controls: mesoporous inorganic-organic hybrid resin (HR4), ECM proteins (fibronectin and collagen IV), and glass. HeLa cell viability on HR4-DOPA was 86.1%, indicating insignificant growth inhibition. We also investigated the optimal microwell depth and cell concentration for HeLa single-cell occupancy: 25-µm-deep microwells at 1.0 × 10^6 cells/ml demonstrated 67.5% single-cell occupancy while providing sufficient cell-adhesive surface area for long-term cell culture (≥ 3000 µm^2). We obtained singly occupied microwells with only 5.9% array-to-array variation, thus providing adequate throughput for accurate quantification in advanced single-cell analyses. Arrays of HR4-DOPA-coated microwells can be used for high-throughput single-cell-based assays for drug discovery as a “bio-cell processor.” Given that the microwell arrays are integrated in a microfluidic biochip, they can mimic the in vivo microenvironment; we can thus predict in vivo responses through high-throughput, isolated single-cell analysis to assess cellular chemistry at the molecular level.In der Medikamentenforschung ist eine schnelle Entscheidungsfindung erforderlich, um neue chemische Stoffe aus einem Kandidaten-Pool möglicher neuer Medikamente zu finden, die eine Chance auf Markteinführung haben. Konventionelle, in vitro, zell-basierte Methoden liefern oft irreführende Ergebnisse, die nicht der in vivo Antwort entsprechen. Wir haben ein Einzelzell Array in einer mikrofluidischen Platform entwickelt, das ein gleichförmiges Millieu für die Zellen bereit stellt, in vivo Funktionalität imitieren kann und biologische Effekte eines Medikaments auf die Chemie einer einzelnen Zelle auf molekularer Ebene demonstrieren kann. Ein dopaminerges, mesopores, inorganisches/organisches Harz (HR4-DOPA) wurde benutzt um die Microwells einer 200-Microwell-Platte zu beschichten. Die Biokompatibilität dieses Harzes wurde durch Zell-Adhäsions- und Viabilitäts-Experimente nachgewiesen. Die Anlagerung von HeLa Zellen an HR4-DOPA war vergleichbar mit der an den Vergleichssubstanzen: mit HR4 (Hybrides, mesoporöses, inorganisches/organisches Harz), mit Proteinen der extrazellulären Matrix (Fibronectin, Collagen IV) und mit biologischem Glas. Die Viabilität der HeLa Zellen auf HR4-DOPA lag dabei bei 86,1%, was auf eine geringe Wachstumshemmung hindeutet. Die optimale Tiefe der Wells und die optimal Anzahl der Zellen wurden ebenfalls untersucht, wobei eine Zellzahl von 1,0 × 106 Zellen/ml und eine Tiefe der Microwells von 25 µm die besten Ergebnisse lieferten mit einer Einzel-Zell Belegung der Wells von 67,5% und eine genügend große Fläche (≥ 3000 µm2) für die Zellanlagerung und Langzeit-Kultivierung bereit stellt. Die Reproduzierbarkeit der Ergebnisse war dabei hoch, lediglich 5,9% Abweichung wurde festgestellt. Somit liefert dieser Ansatz eine geeignete Methode für die exakte Bestimmung und Quantifizierung verschiedener zellulärer Prozesse bei der state-of-the-art Einzel-Zell Analyse. Anordnungen von HR4-DOPA beschichteten Microwells können für Hochdurchsatz Untersuchungen in der Medikamentenforschung als „Bio-Zell Prozessor“ verwendet werden. Vorausgesetzt die Microwells sind integriert in einen mikrofluidischen Biochip, können diese eine in vivo ähnliche Mikroumgebung bereit stellen für weiterführende Versuche. Somit wird es möglich, in vivo Reaktionen akkurat und in einem Hochdurchsatz Verfahren voraus zu sagen und zelluläre Reaktionen auf der molekularen Ebene zu testen

Encoded hydrogel microparticles for high-throughput molecular diagnostics and personalized medicine

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2012.Cataloged from PDF version of thesis.Includes bibliographical references (p. 141-161).The ability to accurately detect and quantify biological molecules in complex mixtures is crucial in basic research as well as in clinical settings. Advancements in genetic analysis, molecular diagnostics, and patient-tailored medicine require robust detection technologies that can obtain high-density information from a range of physiological samples in a rapid and cost-effective manner. Compared to conventional microarrays and methods based on polymerase chain reaction (PCR), suspension (particle-based) arrays offer several advantages in the multiplexed detection of biomolecules, including higher rates of sample processing, reduced consumption of sample and reagent, and rapid probe-set modification for customizable assays. This thesis expands the utility of a novel hydrogel-based microparticle array through (1) the creation of a microfluidic, flow-through fluorescence scanner for high-throughput particle analysis, (2) the development of a suite of techniques for the highly sensitive and specific detection of microRNA (miRNA) biomarkers, and (3) the investigation of new methods for directly measuring biomolecules at the single-cell level. Graphically-encoded hydrogel microparticles synthesized from non-fouling, bioinert poly(ethylene glycol) (PEG) and functionalized with biomolecule probes offer great promise in the development of high-performance, multiplexed bioassays. To extend this platform to applications in high-throughput analysis, particle design was optimized to ensure mechanical stability in high-velocity flow systems, and a single-color microfluidic scanner was constructed for the rapid fluorescence interrogation of each particle's spatially-segregated "code" and "probe" regions. The detection advantages of three-dimensional, probe-laden hydrogel scaffolds and the operational efficiencies of suspension array technology were then leveraged for the rapid multiplexed expression profiling of miRNA. The graphical encoding method and ligationbased labeling scheme implemented here allowed for scalable multiplexing with a simple workflow and an unprecedented combination of sensitivity, flexibility, and throughput. Through the rolling circle amplification of a labeling oligonucleotide, it was possible to further enhance the system's sensitivity and resolve single-molecule miRNA binding events on particle surfaces, enabling the first direct detection of low-abundance miRNA in human serum without the need for RNA extraction or target amplification. Finally, by arraying cells and gel particles in polydimethylsiloxane (PDMS) microwells, it was possible to dramatically improve the particles' target capture efficiency and thereby move closer to a regime in which miRNAs and other biological molecules may be directly detected without target amplification from single cells.by Stephen Clifford Chapin.Ph.D