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ADIPOSE STEM CELLS (ASCs) ISOLATION BY ON-CHIP PRE-TREATMENT OF BIOLOGICAL SAMPLES
Marion Valette, PhD student, LAAS
Adipose stem cells (ASCs) are of considerable interest for regenerative medicine or type II diabetes diagnosis. Migrating and circulating in lymph, their circulation in blood is not excluded[1]. They are supposed to circulate in very small numbers and no method exists to isolate them from blood.
As ASCs do not present specific physical characteristic, we developed a two-steps LOC enabling their isolation by combining size exclusion followed by immuno exclusion. This study presents the first step, based on hydrodynamic filtration, which aims at eliminating biological elements with a diameter below 10µm, corresponding to Red Blood Cells (RBCs), platelets and lymphocytes.
LOC were manufactured using a performing and low cost microfabrication technique based on the lamination and lithography of photosensitive dry films.
Tests were conducted on synthetic solutions of RBCs and ASCs mimicking natural samples. We demonstrated that 100% of ASCs were recovered regardless of the ratio r=[(concentration RBCs)/(concentration ASCs)] but that the filtration yield of RBCs could vary from 45.3% to 55.8% for r ranging from 1.8e2 to 1.3e4. RBC filtration yield could be significantly improved by increasing the number of filtration channel for instance.
Hydrodynamic separation efficiently pre-isolates adipose stem cells from complex samples without damaging cells.
Low-costs, real-time biosensor using loop mediated isothermal amplification (LAMP)
Dogukan Kaygusuz, Author (MSc Student), Sabanci University
Since the beginning of genetically modified organism (GMO) usage, there has been on-going concern and debate about the commercialization of products derived from GMOs. There is an urgent need for developing highly efficient and easy to use methods to carry out rapid and high throughput screening of GMO components and to label GMO-derived foods appropriately. Herein, we present an alternative user-friendly GMO-diagnostic biosensor. Our proposed design is based on a simple gene recognition mechanism; the loop mediated isothermal amplification reaction (LAMP). The LAMP reaction requires 30 minutes under the physical conditions that our printed circuit board (PCB) is provided. The reaction occurs in the PDMS mini-wells and the case of the biosensor is fabricated using 3D printer based on Solid works design. The biosensor allows real-time monitoring of the LAMP reaction thanks to the colorimetric readout of the Hydroxy naphthol blue (HNB) reagent. HNB provides naked-eye visualization of each LAMP reaction under indoor light. Therefore, the GMO positive results are differentiated through a color change from violet to sky blue, while the negative results remain violet. Furthermore, our approach could certainly be modified for detection of other organisms. Last but not least, it is possible to be optimized for high-throughout for professional usage or kept simple for practical operations at home or fields.
A fast and simple contact printing approach to generate 2D Protein Nanopatterns
Veronika Eibl, Researcher, STRATEC Consumables GmbH
Protein micropatterning has become an important tool for many biomedical applications as well as in academic research. Current techniques that allow to reduce the Feature size of patterns below 1µm are, however, often costly and require sophisticated equipment. We present here a straightforward and convenient method to generate
highly condensed nanopatterns of proteins without the need for clean room facilities or expensive equipment. Our approach is based on nanocontact printing and allows to
fabricate protein patterns with feature sizes of 80 nm and periodicities down to 140 nm. This was made possible by the use of the material X-poly(dimethylsiloxane) (X-PDMS)
in a two-layer stamp layout for protein printing. In a proof of principle, different Proteins at various scales were printed and the pattern quality was evaluated by atomic force
microscopy (AFM) and super-resolution fluorescence microscopy.
Cell Counting with Giant Magneto-Resistive Sensors
Manon Giraud, PHD, CEA
The development of rapid, simple, cheap and specific early diagnostic tools, enabling the single-cell detection, is a real challenge in the medical field. The approach proposed in this work relies on a dynamic magnetic detection. Targeted cells are labeled with magnetic beads functionalized with cell-specific antibodies. Then, the immunocaptured cells are injected in a microfluidic channel located above highly sensitive giant magnetoresistance sensors (GMR) with a detectivity of about 100pT/sqrt(Hz) at 1kHz. A perpendicular homogeneous magnetic field polarizes the moving magnetic beads and field variations induced by the resulting stray dipolar field is detected. The first proof of specificity of such a test has been obtained in our lab with NS1 murine myeloma cells. The obtained sensitivity is equivalent to the one of an ELISA test realized on the same biological system. This device is still able to detect every single cell but presently, the sensitivity is limited by false positives created by magnetic particles aggregates flowing very near the sensors which give a signal comparable to cells flowing at larger distances.
Integration and automation of 3D flow-focusing microfluidic system for single particle sorting
Yingkai Lyu, PhD Student, University of Glasgow
The separation and sorting of particles (cells, beads, and droplets) are critical in a variety of biomedical applications including early disease diagnostics [1], therapeutics, cell tracking [2], and clinical research etc. The current standard methods are using 2D microfluidic chips fabricated by PDMS [3]. However, it’s substantially limited to adjust the flow flexibly as the researchers intend to conduct multi-functional assays in a single experiment. Furthermore, the signals detected in the microscale channels tend to be easily deteriorated by the PDMS as well as the substrate materials. This work aimed to develop a 3D integrated and automated microfluidic system with the flexibility and stability for multi-functional assays of different particles. A sorting accuracy higher than 95% was experimentally achieved and reasonably high purity of 85.2% was eventually realized with the initial ratio of the targeted fluorescent beads standing at approximate 17%. The novelty towards this study lies in the 3D flow-focusing function in the detection area, effectively demonstrating the flexibility and stability of the system. Therefore, this method would be promising to apply in a variety of single particle analysis within a highly compatible microfluidic chip.
fluid resistance optimized microfluidic shear device for mechanotransduction studies
Sanat Dash, phd scholar, iit madras
A logarithmic microfluidic shear device was designed and fabricated for cellular mechanotransduction studies. The device contains four cell culture chambers in which flow was modulated to achieve a logarithmic increment. Resistance values were optimized to make the device compact. The network of resistances was developed according to a unique combination of series and parallel resistances as found via optimization. Simulation results done in Ansys 16.1 matched the analytical calculations and showed the shear stress distribution at different inlet flow rates. Fabrication of the device was carried out using conventional photolithography and PDMS soft lithography. Flow profile was validated taking DI water as working fluid and measuring the volume collected at all four outlets. Volumes collected at the outlets were in accordance with the simulation results at inlet flow rates ranging from 1 ml/min to 0.1 ml/min. The device can exert fluid shear stresses ranging four orders of magnitude on the culture chamber walls which will cover shear stress values from interstitial flow to blood flow. This will allow studying cell behavior in the long physiological range of shear stress in a single run reducing number of experiments.
Printable cell culture platform
Diosangeles Soto, Student, Abo Akademi University
Traditional cell culture relies mostly on flat plastic surfaces, such as Petri dishes and multiwall plates. These commercial platforms are difficult to modify, which limits the range of customisation, functionalisation, and monitoring capabilities. The limitations restrict experimental design and expand the gap between in vitro and in vivo research. In contrast, cell behaviour research expands rapidly with the development of microfluidics and electrochemical detection methods. This study proposes a new approach for cell culture: a low-cost device based on stacks of a transparent flexible printable substrate and hydrophobic boundaries. The novelty is the introduction and stacking of layers for potential manipulation and access to the cells. Moreover, the use of a flexible printable film means that the device can be manufactured and modified using conventional coating, printing, and converting techniques. In this research, the concept is demonstrated using human dermal fibroblasts and integrating printed patterns or mineral coatings to influence cell fate. In summary, the device is easy and fast to manufacture, scalable, and can be potentially customised to influence, monitor, and assess cell behaviour. The end-use is not limited to cell culture but can be expanded to other areas, such as drug discovery, diagnostics, point-of-care, and environmental sensing.
Micropillar-assisted electric field enhancement for high-efficiency inactivation of bacteria
Sanam Pudasaini , PhD Student, Nanyang Technological University
Development of high-efficiency and environment friendly bacterial inactivation methods is of great importance for preventing waterborne diseases which are one of the leading causes of death in the world. Traditional bacterial inactivation techniques have several limitations such as longer treatment time, use of harmful chemicals, formation of toxic byproducts, bacterial regrowth etc. Here, an electroporation-based continuous flow insulator-based AC dielectrophoresis device (iDEP) is developed and that the device achieved high bacterial inactivation performance (>99.9%) within a short exposure time (<1 s). Inactivation performance was evaluated for Escherichia coli and Enterococcus faecalis under various electric field conditions. More than 4 log removal of bacteria was obtained with an applied voltage of 300 V for the flowrate of 1 mL/hr. Images from scanning electron microscope confirmed the formation of electroporation-induced nano-pore within the cell membrane. The reported method of inactivation does not involve any chemicals and the formation of harmful by-products is also minimized.
Virtual prototyping and automated lab-on-chip and biosensor simulator for first step design
Alexi BONAMENT, PHD Student, Icube
Recently, lab-on-chips have become an important field of research at the interface between engineering science and biotechnologies. The lab-on-chip is composed of: biochemical reactions, a microfluidic system, biosensors and an electronic circuit for driving, processing and conditioning the signal. Their current technological limits for their industrial development are two types: 1) The absence of standard, reliable and repeatable manufacturing technologies. 2) The inherent complexity of their design, mixing different fields such as biology, microfluidics and electronics.
During the last 10 years, several investigations on the development of CAD tools dedicated to this technology have been reported in the literature have led. Literature work presented mainly revolves around the modelling and simulation of microfluidic circuits using Kirchhoff laws which can then be implemented on an electronic simulator[1]. In lab-on-chip devices, the analysis area is a critical part. To handle this issue, we developed a simulator which takes the geometry of reaction into account[2]. It combines compact models for microfluidics channels (using analogue equivalent circuits), resolution of Navier-Stockes equations at the steady states and a convection-diffusion model for chemical species solved with a finite difference approach. Simulations are performed with SPICE and Python interfaces are built in order to interface the different bricks of the software environment. Our simulator has been validated by comparison with COMSOL simulation results on several use cases.
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