Several biomedical analyses are performed on particular types of cells present in body samples or using functionalized microparticles. Success in such analyses depends on the ability to separate or isolate the target cells or microparticles from the rest of the sample. In conventional procedures, multiple pieces of equipment, such as centrifuges, magnets, and macroscale filters, are used for such purposes, which are time-consuming, associated with human error, and require several operational steps. In the past two decades, there has been a tendency to develop microfluidic techniques, so-called lab-on-a-chip, to miniaturize and automate these procedures. The processes used for the separation and isolation of the cells and microparticles are scaled down into a small microfluidic chip, requiring very small amounts of sample. Differences in the physical and biological properties of the target cells from the other components present in the sample are the key to the development of such microfluidic techniques. These techniques are categorized as filtration-, hydrodynamic-, dielectrophoretic-, acoustic- and magnetic-based methods. Here we review the microfluidic techniques developed for sorting, separation, and isolation of cells and microparticles for biomedical applications. The mechanisms behind such techniques are thoroughly explained and the applications in which these techniques have been adopted are reviewed.

Rare cells in blood, such as circulating tumor cells or fetal cells in the maternal circulation, posses a great prognostic or diagnostic value, or for the development of personalized medicine, where the study of rare cells could provide information to more specifically targeted treatments. When conventional cell separation methods, such as flow cytometry or magnetic activated cell sorting, have fallen short other methods are desperately sought for. Microfluidics have been extensively used towards isolating and processing rare cells as it offers possibilities not present in the conventional systems. Furthermore, microfluidic methods offer new possibilities for cell separation as they often rely on non-traditional biomarkers and intrinsic cell properties. This offers the possibility to isolate cell populations that would otherwise not be targeted using conventional methods. Here, we provide an extensive review of the latest advances in continuous flow microfluidic rare cell separation and processing with each cell's specific characteristics and separation challenges as a point of view.

Cancer is one of the foremost causes of death globally. Late-stage presentation, inaccessible diagnosis, and treatment are common challenges in developed countries. Detection, enumeration of Circulating Tumor Cells (CTC) as early as possible can reportedly lead to more effective treatment. The isolation of CTC at an early stage is challenging due to the low probability of its presence in peripheral blood. In this study, we propose a novel two-stage, label-free, rapid, and continuous CTC separation device based on hydrodynamic inertial focusing and dielectrophoretic separation. The dominance and differential of wall-induced inertial lift force and Dean drag force inside a curved microfluidic channel results in size-based separation of Red Blood Cells (RBC) and platelets (size between 2-4 µm) from CTC and leukocytes (9-12.2 µm). A numerical model was used to investigate the mechanism of hydrodynamic inertial focusing in a curvilinear microchannel. Simulations were done with the RBCs, platelets, CTCs, and leukocytes (four major subtypes) to select the optimized value of the parameters in the proposed design. In first stage, the focusing behavior of microscale cells was studied to sort leukocytes and CTCs from RBCs, and platelets while viable CTCs were separated from leukocytes based on their inherent electrical properties using dielectrophoresis in the second stage. The proposed design of the device was evaluated for CTC separation efficiency using numerical simulations. This study considered the influence of critical factors like aspect ratio, dielectrophoretic force, channel size, flow rate, separation efficiency, and shape on cell separation. Results show that the proposed device yields viable CTC with 99.5% isolation efficiency with a throughput of 12.2 ml/h.

The separation of circulating tumor cells (CTCs) from the peripheral blood is an important issue that has been highlighted because of their high clinical potential. However, techniques that depend solely on tumor-specific surface molecules or just the larger size of CTCs are limited by tumor heterogeneity. Here, we present a slanted weir microfluidic device that utilizes the size and deformability of CTCs to separate them from the unprocessed whole blood. By testing its ability using a highly invasive breast cancer cell line, our device achieved a 97% separation efficiency, while showing an 8-log depletion of erythrocytes and 5.6-log depletion of leukocytes. We also developed an image analysis tool that was able to characterize the various morphologies and differing deformability of the separating cells. From the results, we believe our system possesses a high potential for liquid biopsy, aiding future cancer research.

The isolation of a specific lymphocyte subset from blood is the required first step in the manufacturing of many novel cellular immunotherapies. Microfluidic size-based separation methods are poised to significantly simplify this process because they require neither centrifugation nor magnetic or fluorescent labeling to operate. Lymphocytes can be separated from red blood cells (RBCs) and platelets as well as monocytes and granulocytes because their size differs from each of these cell types. However, further separation of a specific lymphocyte subset from other unwanted lymphocytes using size-based methods is impossible because all lymphocytes have approximately the same size and can only be distinguished by surface markers. This paper describes a new approach that made it possible for a size-based separation method to isolate a desired subset of lymphocytes by making unwanted lymphocytes as well as other blood cells artificially larger. The separation was enabled by selectively binding multiple RBCs to each unwanted cell to create 'rosettes' with an effective size significantly larger than the diameter of a typical lymphocyte. The desired lymphocytes remained unaffected by rosetting and were separated from the rosettes by passing the mixture through a microfluidic size-based separation device based on controlled incremental filtration (CIF). This new rosette-enabled size-based (RESIZE) separation approach demonstrated recovery of 80-90% for all lymphocyte subsets tested (CD3, CD4, CD56) which was ∼2.5-fold higher than that for the standard immunodensity method (RBC rosetting followed by density gradient centrifugation). The purity of separation was >90% for CD3 cells but declined with increasing cell rarity. Unlike the immunodensity approach, RESIZE required neither centrifugation nor cell washing after the separation and was ∼2.5-fold faster when processing the same sample volume. The results of this study suggest that integration of the RESIZE approach for high-yield isolation of lymphocyte subsets from blood could significantly streamline the manufacturing workflow and thus have a potentially transformative impact on the cost and availability of novel cellular immunotherapies.

Blood is a complex sample comprised mostly of plasma, red blood cells (RBCs), and other cells whose concentrations correlate to physiological or pathological health conditions. There are also many blood-circulating biomarkers, such as circulating tumor cells (CTCs) and various pathogens, that can be used as measurands to diagnose certain diseases. Microfluidic devices are attractive analytical tools for separating blood components in point-of-care (POC) applications. These platforms have the potential advantage of, among other features, being compact and portable. These features can eventually be exploited in clinics and rapid tests performed in households and low-income scenarios. Microfluidic systems have the added benefit of only needing small volumes of blood drawn from patients (from nanoliters to milliliters) while integrating (within the devices) the steps required before detecting analytes. Hence, these systems will reduce the associated costs of purifying blood components of interest (e.g., specific groups of cells or blood biomarkers) for studying and quantifying collected blood fractions. The microfluidic blood separation field has grown since the 2000s, and important advances have been reported in the last few years. Nonetheless, real POC microfluidic blood separation platforms are still elusive. A widespread consensus on what key figures of merit should be reported to assess the quality and yield of these platforms has not been achieved. Knowing what parameters should be reported for microfluidic blood separations will help achieve that consensus and establish a clear road map to promote further commercialization of these devices and attain real POC applications. This review provides an overview of the separation techniques currently used to separate blood components for higher throughput separations (number of cells or particles per minute). We present a summary of the critical parameters that should be considered when designing such devices and the figures of merit that should be explicitly reported when presenting a device's separation capabilities. Ultimately, reporting the relevant figures of merit will benefit this growing community and help pave the road toward commercialization of these microfluidic systems.

A microfluidic device is presented for the continuous separation of red blood cells (RBCs) and white blood cells (WBCs) in a label-free manner based on negative dielectrophoresis (n-DEP). An alteration of the electric field, generated by pairs of slanted electrodes (separators) that is fabricated by covering parts of single slanted electrodes with an insulating layer is used to separate cells by their sizes. The repulsive force of n-DEP formed by slanted electrodes prepared on both the top and bottom substrates led to the deflection of the cell flow in lateral directions. The presence of gaps covered with an insulating layer for the electric field on the electrodes allows the passing of RBCs through gaps, while relatively large WBCs (cultured cultured human acute monocytic leukemia cell line (THP-1 cells)) flowed along the slanted separator without passing through the gaps and arrived at an edge in the channel. The passage efficiency for RBCs through the gaps and the arrival efficiency for THP-1 cells to the upper edge in the channel were estimated and found to be 91% and 93%, respectively.