Singapore-MIT Alliance for Research & Technology

BioSystems and Micromechanics (BioSyM) Inter-Disciplinary Research Group

BioSyM's recent publications in "Lab on A Chip" journal

SMART BioSyM researchers have recently published some of their work in the prominent journal "Lab on A Chip". The articles address 3 applications of microfluidics. The research work described are results of collaborative efforts between researchers of SMART BioSyM, NUS and MIT.

In one titled "Pinched flow coupled shear-modulated inertial microfluidics for high-throughput rare blood cell separation" (Ali Asgar S. Bhagat, Han Wei Hou, Leon D. Li, Chwee Teck Lim and Jongyoon Han, Lab on A Chip, Published on 19 April 2011 on http://pubs.rsc.org | doi:10.1039), the authors describe the application of shear-modulated inertial microfluidics to isolate Circulating Tumor Cells (CTC) from blood. Blood is a complex suspension of cells (40–45% of blood volume) in plasma which plays several key roles including transport of oxygen and nutrients to cells, removal of cellular waste products and providing immunological protection. Red blood cells (RBCs) account for >99% of all hematologic cellular components (5 billion RBCs per millilitre of whole blood) with the remaining <1% consisting of peripheral blood leukocytes (PBL) and platelets. In addition to RBCs and leukocytes, other low abundance cells such as fetal cells, circulating tumor cells (CTCs), stem cells or leukemic cells are also found in peripheral blood of patients, which can potentially be used for various biomedical applications such as disease detection, diagnosis, prognosis, therapeutic treatment monitoring and conducting fundamental scientific studies. However, as the low abundance cells are so rare, it is often necessary to have an enrichment or separation step to efficiently isolate them from blood prior to analysis. This paper describes the application of shear-modulated inertial microfluidics to isolate Circulating Tumor Cells (CTC) from blood.

Schematic illustration of the developed microfluidic device for rare cell isolation from blood. The microchannel design consists of a high aspect ratio rectangular microchannel patterned with a contraction–expansion array. In the cell-focusing region, under the influence of shearmodulated inertial lift forces all the cells equilibrate efficiently along the channel sidewalls. Flowing through the rare cell pinching region, the center of mass of the larger cells are aligned along the channel center while the smaller hematologic cells remain focused along the channel sidewalls. Designing bifurcating outlets allows for the collection of the larger rare cells at the center outlet while the remaining hematologic cells are removed from the side outlets.

Another paper on "High-throughput cell cycle synchronization using inertial forces in spiral microchannels" (Wong Cheng Lee, Ali Asgar S. Bhagat, Sha Huang, Krystyn J. Van Vliet, Jongyoon Han and Chwee Teck Lim, Lab on A Chip, 11, 1359 (2011)), describes a microfluidics based approach to synchronize cells using inertial forces in spiral microchannels. Cell cycle synchronization is essential for studying cellular properties, biological processes and elucidating genetic regulatory mechanisms and events involved in each phase prior to cell division. In eukaryotic cells, the distinct events leading to proper cell division can be separated into four sequential phases: G1(gap), S (DNA synthesis), G2 (gap) and M (mitosis). As a cell progresses through the cell cycle, it duplicates its hromosomes during the S phase and segregates the chromosomes in the M phase. The use of a highly synchronized population of cells has facilitated the development of a variety of biological systems. For example, via such synchronization of cancer cells, several key oncogenes have been identified and implicated in specific cell cycle checkpoints. Development of cancer therapeutics has thus employed tumor cell synchronization, because anticancer drugs are known to target cells in different phases of the cell cycle. In stem cell therapies that involve nuclear transfer to the host cells, cell cycle synchronization is critical because stem cells in the G0/G1 phase impart higher nuclear transfer efficiency. Thus, there is great interest in developing efficient techniques to rapidly synchronize and to isolate cells at various phases of the cell cycle. This paper describes a microfluidics based approach to synchronize cells using inertial forces in spiral microchannels.

	(A) Schematic illustration of the spiral microfluidic design developed for cell cycle synchronization. Under the influence of inertial lift forces and Dean drag force, asynchronous cell populations are size fractionated to obtain relatively pure populations of cells in the G0/G1, S and G2/M phase. The cells in the G2/M phase, due to the larger diameter at this cell cycle stage, equilibrate closest to the microchannel inner wall followed by cells in the S and the G0/G1 phase. Inset: photograph of the spiral microchannel with one inlet and eight outlets fabricated in PDMS. (B) Validation of design principle using fluorescently labeled polystyrene particles. Superimposed images illustrating distribution and position of the 10 mm, 15 mm, and 25 mmdiameter particles at the inlet, a 500 mm wide channel section prior to the outlet, and the bifurcated outlet of a 140 mm high microchannel at 2.5 mL / min flowrate. The randomly distributed particles at the inlet form ordered focused streams which are then collected separately at outlets 1 (largest), 2 and 3 (smallest).
(A) Optical micrographs of the unsorted (control) and sorted hMSCs cells collected from outlets 1, 2, 3 and 4. The mean cell diameter collected at outlet 1 is ~24 um, as compared to ~15 um collected at outlet 4 (p < 0.001). (B) Trypan-blue exclusion by collected cells indicates viability of hMSCs post sorting (arrows indicate non-viable cells). Results indicate that the high shear experienced by the cells in these microchannels do not compromise their viability, achieving >90% cell recovery. (C) Optical micrograph of the re-seeded cells indicating no significant difference between the proliferation rates of cells collected from the outlets as compared to the unsorted population; this indicates high viability and sterility. Scale bar = 50 um.

In the 3rd paper, "A microfluidic system with optical laser tweezers to study mechanotransduction and focal adhesion recruitment" (Peyman Honarmandi, Hyungsuk Lee, Matthew J. Lang and Roger D. Kamm, Lab on A Chip, 11, 684 (2011)), a microfluidic device and optical laser tweezers are used to apply mechanical stimuli to cells. In the last decade, several experiments have investigated the importance of mechanosensing and the assembly of focal complexes at adhesion sites under the application of mechanical force with various ECM proteins. These studies demonstrated that mechanical forces are required for the initiation and
formation of focal complexes through a process termed mechanotransduction. Detailed studies are difficult, however, due to the challenges of applying precise levels of tensile force to the cell under closely regulated conditions. Although more than 40 proteins are known to be present in FA domains, it is not known how many of these are implicated in mechanotransduction. New methods are therefore needed to systematically examine these and their localization to a FA, or other adhesion complexes, under the application of force.

Using microfluidics provides convenient means of introducing and positioning cells under controlled and physiological conditions. In this particular instance, a microfluidic device was designed to confine the cells and to expose a portion of the cell membrane to a bead functionalized with an integrin-binding protein, e.g. fibronectin, to emulate ECM-integrin-cytoskeleton linkages, as illustrated below. An optical laser trap is used to facilitate and apply force in a defined direction on the exposed portion of the cell membrane via its receptors. Then, utilizing immunostaining for proteins of interest, i.e. vinculin in our case, the recruitment of protein is studied upon application of an external tensile load. In addition, the mechanical properties of the FA complex can be inferred from the measurement. The main advantages of the proposed method are controllability, physiological environment, introducing beads, ease of numerical simulations, local excitation with defined geometry, and most importantly the capability to directly image protein recruitment, which other techniques such as atomic force microscopy or micropipette aspiration may not offer.

Schematic representation of activated integrin and formation of ECM-integrin-cytoskeleton linkages in focal adhesion site upon application of an external tensile load.
(a) 3D-schematic representation of microfluidic device with upward patterned surface. A circular coverslip glass is attached on top, (b) Different resolutions of device and channels after fabrication. Channels are filled with food coloring for better visualization.