Circulating tumor cells (CTCs) are important targets for cancer biology studies. To further elucidate the role of CTCs in cancer metastasis and prognosis, effective methods for isolating extremely rare tumor cells from peripheral blood must be developed. Acousticbased methods, which are known to preserve the integrity, functionality, and viability of biological cells using label-free and contactfree sorting, have thus far not been successfully developed to isolate rare CTCs using clinical samples from cancer patients owing to technical constraints, insufficient throughput, and lack of long-term device stability. In this work, we demonstrate the development of an acoustic-based microfluidic device that is capable of high-throughput separation of CTCs from peripheral blood samples obtained from cancer patients. Our method uses tilted-angle standing surface acoustic waves. Parametric numerical simulations were performed to design optimum device geometry, tilt angle, and cell throughput that is more than 20 times higher than previously possible for such devices. We first validated the capability of this device by successfully separating low concentrations (∼100 cells/mL) of a variety of cancer cells from cell culture lines from WBCs with a recovery rate better than 83%. We then demonstrated the isolation of CTCs in blood samples obtained from patients with breast cancer. Our acoustic-based separation method thus offers the potential to serve as an invaluable supplemental tool in cancer research, diagnostics, drug efficacy assessment, and therapeutics owing to its excellent biocompatibility, simple design, and label-free automated operation while offering the capability to isolate rare CTCs in a viable state.circulating cancer cells | cell separation | rare-cell sorting | acoustic tweezers | microfluidics

Separation of cells is a critical process for studying cell properties, disease diagnostics, and therapeutics. Cell sorting by acoustic waves offers a means to separate cells on the basis of their size and physical properties in a label-free, contactless, and biocompatible manner. The separation sensitivity and efficiency of currently available acousticbased approaches, however, are limited, thereby restricting their widespread application in research and health diagnostics. In this work, we introduce a unique configuration of tilted-angle standing surface acoustic waves (taSSAW), which are oriented at an optimally designed inclination to the flow direction in the microfluidic channel. We demonstrate that this design significantly improves the efficiency and sensitivity of acoustic separation techniques. To optimize our device design, we carried out systematic simulations of cell trajectories, matching closely with experimental results. Using numerically optimized design of taSSAW, we successfully separated 2-and 10-μm-diameter polystyrene beads with a separation efficiency of ∼99%, and separated 7.3-and 9.9-μm-polystyrene beads with an efficiency of ∼97%. We illustrate that taSSAW is capable of effectively separating particles-cells of approximately the same size and density but different compressibility. Finally, we demonstrate the effectiveness of the present technique for biological-biomedical applications by sorting MCF-7 human breast cancer cells from nonmalignant leukocytes, while preserving the integrity of the separated cells. The method introduced here thus offers a unique route for separating circulating tumor cells, and for label-free cell separation with potential applications in biological research, disease diagnostics, and clinical practice.particle separation | microfluidics | cancer cell separation | acoustofluidics | tilt-angle optimization

Red blood cells (RBCs) can be cleared from circulation when alterations in their size, shape, and deformability are detected. This function is modulated by the spleen-specific structure of the interendothelial slit (IES). Here, we present a unique physiological framework for development of prognostic markers in RBC diseases by quantifying biophysical limits for RBCs to pass through the IES, using computational simulations based on dissipative particle dynamics. The results show that the spleen selects RBCs for continued circulation based on their geometry, consistent with prior in vivo observations. A companion analysis provides critical bounds relating surface area and volume for healthy RBCs beyond which the RBCs fail the “physical fitness test” to pass through the IES, supporting independent experiments. Our results suggest that the spleen plays an important role in determining distributions of size and shape of healthy RBCs. Because mechanical retention of infected RBC impacts malaria pathogenesis, we studied key biophysical parameters for RBCs infected with Plasmodium falciparum as they cross the IES. In agreement with experimental results, surface area loss of an infected RBC is found to be a more important determinant of splenic retention than its membrane stiffness. The simulations provide insights into the effects of pressure gradient across the IES on RBC retention. By providing quantitative biophysical limits for RBCs to pass through the IES, the narrowest circulatory bottleneck in the spleen, our results offer a broad approach for developing quantitative markers for diseases such as hereditary spherocytosis, thalassemia, and malaria.

We study the biomechanical interactions between the lipid bilayer and the cytoskeleton in a red blood cell (RBC) by developing a general framework for mesoscopic simulations. We treated the lipid bilayer and the cytoskeleton as two distinct components and developed a unique whole-cell model of the RBC, using dissipative particle dynamics (DPD). The model is validated by comparing the predicted results with measurements from four different and independent experiments. First, we simulated the micropipette aspiration and quantified the cytoskeletal deformation. Second, we studied the membrane fluctuations of healthy RBCs and RBCs parasitized to different intraerythrocytic stages by the malaria-inducing parasite Plasmodium falciparum. Third, we subjected the RBC to shear flow and investigated the dependence of its tank-treading frequency on shear rate. Finally, we simulated the bilayer-cytoskeletal detachment in channel flow to quantify the strength of such interactions when the corresponding bonds break. Taken together, these experiments and corresponding systematic DPD simulations probe the governing constitutive response of the cytoskeleton, elastic stiffness, viscous friction, and strength of bilayer-cytoskeletal interactions as well as membrane viscosities. Hence, the DPD simulations and comparisons with available independent experiments serve as validation of the unique two-component model and lead to useful insights into the biomechanical interactions between the lipid bilayer and the cytoskeleton of the RBC. Furthermore, they provide a basis for further studies to probe cell mechanistic processes in health and disease in a manner that guides the design and interpretation of experiments and to develop simulations of phenomena that cannot be studied systematically by experiments alone.coarse graining | worm-like chain | multiscale modeling | adhesion energy | erythrocyte T he red blood cell (RBC) membrane consists of two components: a lipid bilayer and an attached 2D spectrin network that acts as the cytoskeleton. The resistance of the lipid bilayer to bending is controlled by the bending rigidity, k c , whereas the spectrin network's resistance to shear strain is characterized by the in-plane shear modulus, μ s . Under normal conditions, the cytoskeleton is tightly attached to the lipid bilayer from the cytoplasmic side. However, under certain pathological conditions, e.g., in sickle cell disease, the cytoskeleton may become dissociated from the lipid bilayer (1). Although the biomechanics of the two-component erythrocyte membrane have been studied extensively for decades (2), the mechanical properties of the interactions between the lipid bilayer and the cytoskeleton (such as elastic stiffness, viscous friction, and strength) via the pinning connections of transmembrane proteins are still largely unknown. This is at least in part ascribed to the fact that it is difficult to measure these interactions directly from experiments, because the length scale of these connections is too small compared with the chara...

The proposed method is fully automatic without any user interaction. Quantitative results reveal that the proposed approach is efficient and accurate for hepatic volume estimation in a clinical setup. The high correlation between the automatic and manual references shows that the proposed method can be good enough to replace the time-consuming and nonreproducible manual segmentation method.

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To quantitatively predict the mechanical response and mechanically induced remodeling of red blood cells, we developed a multiscale method to correlate distributions of internal stress with overall cell deformation. This method consists of three models at different length scales: in the complete cell level the membrane is modeled as two distinct layers of continuum shells using finite element method (Level III), in which the skeleton-bilayer interactions are depicted as a slide in the lateral (i.e. in-plane) direction (caused by the mobility of the skeleton-bilayer pinning points) and a normal contact force; the constitutive laws of the inner layer (the protein skeleton) are obtained from a molecular-based model (Level II); the mechanical properties of the spectrin (Sp, a key component of the skeleton), including its folding/unfolding reactions, are obtained with a stress-strain model (Level I). Model verification is achieved through comparisons with existing numerical and experimental studies in terms of the resting shape of the cell as well as cell deformations induced by micropipettes and optical tweezers. Detailed distributions of the interaction force between the lipid bilayer and the skeleton that may cause their dissociation and lead to phenomena such as vesiculation are predicted. Specifically, our model predicts correlation between the occurrence of Sp unfolding and increase in the mechanical load upon individual skeleton-bilayer pinning points. Finally a simulation of the necking process after skeleton-bilayer dissociation, a precursor of vesiculation, is conducted.

Summary Gametocyte maturation in Plasmodium falciparum is a critical step in the transmission of malaria. While the majority of parasites proliferate asexually in red blood cells, a small fraction of parasites undergo sexual conversion and mature over 2 weeks to become competent for transmission to a mosquito vector. Immature gametocytes sequester in deep tissues while mature stages must be able to circulate, pass the spleen and present themselves to the mosquito vector in order to complete transmission. Sequestration of asexual red blood cell stage parasites has been investigated in great detail. These studies have demonstrated that induction of cytoadherence properties through specific receptor–ligand interactions coincides with a significant increase in host cell stiffness. In contrast, the adherence and biophysical properties of gametocyte‐infected red blood cells have not been studied systematically. Utilizing a transgenic line for 3D live imaging, in vitro capillary assays and 3D finite element whole cell modelling, we studied the role of cellular deformability in determining the circulatory characteristics of gametocytes. Our analysis shows that the red blood cell deformability of immature gametocytes displays an overall decrease followed by rapid restoration in mature gametocytes. Intriguingly, simulations suggest that along with deformability variations, the morphological changes of the parasite may play an important role in tissue distribution in vivo. Taken together, we present a model, which suggests that mature but not immature gametocytes circulate in the peripheral blood for uptake in the mosquito blood meal and transmission to another human host thus ensuring long‐term survival of the parasite.