Microfluidic inertia enhanced phase partitioning for enriching nucleated cell populations in blood.

Nucleated cells in blood like white blood cells (WBCs) and other rare cells including peripheral blood stem cells (PBSCs) and circulating tumor cells (CTCs) possess significant value for patient monitoring and clinical diagnosis. Enrichment of nucleated cells from contaminating red blood cells (RBCs) using label-free techniques without the use of antibodies or centrifugation is highly desirable to ensure minimal cell loss and activation. To accomplish this, we demonstrate proof-of-concept of a new microfluidic technique that combines aqueous phase partitioning with inertial focusing to accomplish enrichment of nucleated cells in blood. This technique exploits selective affinity of RBCs to the dextran phase (DEX) to accomplish initial separation which is amplified by inertial forces that develop in high-aspect-ratio channels. In our experiments, we spiked RBC samples with representative nucleated cells, MOLT-3 cells (human, peripheral blood, T lymphoblast cell line) and MCF-7 cells (human breast cancer cell line) in a ratio of 500 : 1 (RBCs : nucleated cells) and accomplished depletion of ~96% of RBCs while retaining ~98% of nucleated cells. Higher purity can be accomplished by subjecting the enriched nucleated cell mixture to a second pass via the same process. The second pass further enhances RBC depletion (>99% of initial concentration) whereas nucleated cells were recovered without any further loss. This technique therefore has the potential to be utilized either alone or as a sample preparation tool in the clinical and research setting for various clinical and research applications.

Inertial lift enhanced phase partitioning for continuous microfluidic surface energy based sorting of particles.

A new microfluidics technique that exploits the selectivity of phase partitioning and high-speed focusing capabilities of the inertial effects in flow was developed for continuous label-free sorting of particles and cells. Separations were accomplished by introducing particles at the interface of polyethylene glycol (PEG) and dextran (DEX) phases in rectangular high aspect-ratio microfluidic channels and allowing them to partition to energetically favorable locations within the PEG phase, DEX phase or interface at the center of the microchannel. Separation of partitioned particles was further enhanced via inertial lift forces that develop in high aspect-ratio microchannels that move particles to equilibrium positions close to the outer wall. Combining phase partitioning with inertial focusing ensures selectivity is possible using phase partitioning with sufficient throughput (at least an order of magnitude greater than phase partitioning alone) for application in the clinical and research setting. Using this system we accomplished separation of 15 µm polystyrene (PS) particles from 1-20 µm polymethylmethacrylate (PMMA) particles. Results confirm the feasibility of separation based on phase partitioning and enhancement of separation via inertial focusing. Approximately 86% of PS particles were isolated within the PEG phase whereas 78% of PMMA particles were isolated within the DEX phase. When a binary mixture of PS and PMMA was introduced within the device, ~83% of PS particles were isolated in the PEG phase and ~74% of PMMA particles were isolated in the DEX phase. These results confirm the feasibility of this technique for rapid and reliable separation of particles and potentially cells.

Endothelial cell culture model for replication of physiological profiles of pressure, flow, stretch, and shear stress in vitro.

The phenotype and function of vascular cells in vivo are influenced by complex mechanical signals generated by pulsatile hemodynamic loading. Physiologically relevant in vitro studies of vascular cells therefore require realistic environments where in vivo mechanical loading conditions can be accurately reproduced. To accomplish a realistic in vivo-like loading environment, we designed and fabricated an Endothelial Cell Culture Model (ECCM) to generate physiological pressure, stretch, and shear stress profiles associated with normal and pathological cardiac flow states. Cells within this system were cultured on a stretchable, thin (∼500 µm) planar membrane within a rectangular flow channel and subject to constant fluid flow. Under pressure, the thin planar membrane assumed a concave shape, representing a segment of the blood vessel wall. Pulsatility was introduced using a programmable pneumatically controlled collapsible chamber. Human aortic endothelial cells (HAECs) were cultured within this system under normal conditions and compared to HAECs cultured under static and "flow only" (13 dyn/cm(2)) control conditions using microscopy. Cells cultured within the ECCM were larger than both controls and assumed an ellipsoidal shape. In contrast to static control control cells, ECCM-cultured cells exhibited alignment of cytoskeletal actin filaments and high and continuous expression levels of ß-catenin indicating an in vivo-like phenotype. In conclusion, design, fabrication, testing, and validation of the ECCM for culture of ECs under realistic pressure, flow, strain, and shear loading seen in normal and pathological conditions was accomplished. The ECCM therefore is an enabling technology that allows for study of ECs under physiologically relevant biomechanical loading conditions in vitro.

Exploiting osmosis for blood cell sorting.

Blood is a valuable tissue containing cellular populations rich in information regarding the immediate immune and inflammatory status of the body. Blood leukocytes or white blood cells (WBCs) provide an ideal sample to monitor systemic changes and understand molecular signaling mechanisms in disease processes. Blood samples need to be processed to deplete contaminating erythrocytes or red blood cells (RBCs) and sorted into different WBC sub-populations prior to analysis. This is typically accomplished using immuno-affinity protocols which result in undesirable activation. An alternative is size based sorting which by itself is unsuitable for WBCs sorting due to size overlap between different sub-populations. To overcome this limitation, we investigated the possibility of using controlled osmotic exposure to deplete and/or create a differential size increase between WBC populations. Using a new microfluidic cell docking platform, the response of RBCs and WBCs to deionized (DI) water was evaluated. Time lapse microscopy confirms depletion of RBCs within 15 s and creation of > 3 µm size difference between lymphocytes, monocytes and granulocytes. A flow through microfluidic device was also used to expose different WBCs to DI water for 30, 60 and 90 s to quantify cell loss and activation. Results confirm preservation of ~100% of monocytes, granulocytes and loss of ~30% of lymphocytes (mostly CD3+/CD4+) with minimal activation. These results indicate feasibility of this approach for monocyte, granulocyte and lymphocyte (sub-populations) isolation based on size.

Microfluidic cardiac cell culture model (µCCCM).

Physiological heart development and cardiac function rely on the response of cardiac cells to mechanical stress during hemodynamic loading and unloading. These stresses, especially if sustained, can induce changes in cell structure, contractile function, and gene expression. Current cell culture techniques commonly fail to adequately replicate physical loading observed in the native heart. Therefore, there is a need for physiologically relevant in vitro models that recreate mechanical loading conditions seen in both normal and pathological conditions. To fulfill this need, we have developed a microfluidic cardiac cell culture model (µCCCM) that for the first time allows in vitro hemodynamic stimulation of cardiomyocytes by directly coupling cell structure and function with fluid induced loading. Cells are cultured in a small (1 cm diameter) cell culture chamber on a thin flexible silicone membrane. Integrating the cell culture chamber with a pump, collapsible pulsatile valve and an adjustable resistance element (hemostatic valve) in series allow replication of various loading conditions experienced in the heart. This paper details the design, modeling, fabrication and characterization of fluid flow, pressure and stretch generated at various frequencies to mimic hemodynamic conditions associated with the normal and failing heart. Proof-of-concept studies demonstrate successful culture of an embryonic cardiomyoblast line (H9c2 cells) and establishment of an in vivo like phenotype within this system.