Rapid, label-free enrichment of lymphocytes in a closed system using a flow-through microfluidic device.

The majority of adoptive cellular therapies are produced from peripheral mononuclear cells obtained via leukapheresis and further enriched for the cells of interest (e.g., T cells). Here, we present a first-of-its-kind closed system, which effectively removes ~85% of monocytes and ~88% of platelets, while recovering ~88% of concentrated T cells in a separate output stream, as the leukapheresis sample flows through a microfluidic device at 5 mL/min. The system is driven by a common peristaltic pump, enabled by a novel pressure wave dampener, and operates in a closed bag-to-bag configuration, without requiring any specialized, dedicated equipment. When compared to standard density gradient centrifugation on paired samples, the new system demonstrated a 1.5-fold increase in T cell recovery and a 2-fold reduction in inter-sample variability for this separation outcome. The T cell-to-monocyte ratio of the leukapheresis sample was increased to 20:1, whereas with density gradient processing it decreased to 2:1. As a result of superior purity and/or gentler processing, T cells enriched by the system showed a 2.7-times higher fold expansion during subsequent culture, and an overall 3.5-times higher cumulative yield. This centrifugation-free and label-free closed system for enriching lymphocytes could significantly simplify and standardize the manufacturing of life-saving cellular therapies.

Separation of platelets by size in a microfluidic device based on controlled incremental filtration.

The significant biological and functional differences between small and large platelets suggested by recent studies could have profound implications for transfusion medicine. However, investigating the relationship between platelet size and function is challenging because separating platelets by size without affecting their properties is difficult. A standard approach is centrifugation, but it inevitably leads to premature activation and aggregation of separated platelets. This paper describes the development and validation of a microfluidic device based on controlled incremental filtration (CIF) for separating platelets by size without the cell damage and usability limitations associated with centrifugation. Platelet samples derived from whole blood were used to evaluate the dependence of the CIF device separation performance on design parameters and flow rate, and to compare the properties of PLT fractions generated by the CIF device with those produced using a centrifugation protocol in a split-sample study. This was accomplished by quantifying the platelet size distribution, mean platelet volume (MPV), platelet-large cell ratio (P-LCR) and platelet activation before and after processing for all input and output samples. The 'large platelet' fractions produced by the CIF device and the centrifugation protocol were essentially equivalent (no significant difference in MPV and P-LCR). Platelets in the 'small platelet' fraction produced by the CIF device were significantly smaller than those produced by centrifugation (lower MPV and P-LCR). This was because the CIF 'small platelet' fraction was contaminated by much fewer large platelets (∼2-times lower recovery of >12 fL platelets) and retained the smallest platelets that were discarded by the centrifugation protocol. There was no significant difference in platelet activation between the two methods. However, centrifugation required a substantial amount of additional anticoagulant to prevent platelet aggregation during pelleting. Unlike centrifugation, the CIF device offered continuous, flow-through, single-step processing that did not cause platelet aggregation. Such a capability has the potential to accelerate the basic studies of the relationship between platelet size and function, and ultimately improve transfusion practice, particularly in the pediatric setting, where the need for low-volume, high-quality platelet transfusions is most urgent.

Red blood cell rosetting enables size-based separation of specific lymphocyte subsets from blood in a microfluidic device.

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.

Improved expansion of T cells in culture when isolated with an equipment-free, high-throughput, flow-through microfluidic module versus traditional density gradient centrifugation.

BACKGROUND: The isolation of lymphocytes - and removal of platelets (PLTs) and red blood cells (RBCs) - from an initial blood sample prior to culture is a key enabling step for effective manufacture of cellular therapies. Unfortunately, currently available methods suffer from various drawbacks, including low cell recovery, need for complex equipment, potential loss of sterility and/or high materials/labor cost. METHODS: A newly developed system for selectively concentrating leukocytes within precisely designed, but readily fabricated, microchannels was compared with conventional density gradient centrifugation with respect to: (i) ability to recover lymphocytes while removing PLTs/RBCs and (ii) growth rate and overall cell yield once expanded in culture. RESULTS: In the optimal embodiment of the new microfluidic approach, recoveries of CD3+, CD19+ and CD56+ cells (85%, 89% and 97%, respectively) were significantly higher than for paired samples processed via gradient-based separation (51%, 53% and 40%). Although the removal of residual PLTs and RBCs was lower using the new approach, its enriched T-cell fraction nevertheless grew at a significantly higher rate than the gradient-isolated cells, with approximately twice the cumulative cell yield observed after 7 days of culture. DISCUSSION: The standardization of each step of cellular therapy manufacturing would enable an accelerated translation of research breakthroughs into widely available clinical treatments. The high-throughput approach described in this study - requiring no ancillary pumping mechanism nor expensive disposables to operate - may be a viable candidate to standardize and streamline the initial isolation of lymphocytes for culture while also potentially shortening the time required for their expansion into a therapeutic dose.

A portable system for processing donated whole blood into high quality components without centrifugation.

BACKGROUND: The use of centrifugation-based approaches for processing donated blood into components is routine in the industrialized world, as disparate storage conditions require the rapid separation of 'whole blood' into distinct red blood cell (RBC), platelet, and plasma products. However, the logistical complications and potential cellular damage associated with centrifugation/apheresis manufacturing of blood products are well documented. The objective of this study was to evaluate a proof-of-concept system for whole blood processing, which does not employ electromechanical parts, is easily portable, and can be operated immediately after donation with minimal human labor. METHODS AND FINDINGS: In a split-unit study (n = 6), full (~500mL) units of freshly-donated whole blood were divided, with one half processed by conventional centrifugation techniques and the other with the new blood separation system. Each of these processes took 2-3 hours to complete and were performed in parallel. Blood products generated by the two approaches were compared using an extensive panel of cellular and plasma quality metrics. Comparison of nearly all RBC parameters showed no significant differences between the two approaches, although the portable system generated RBC units with a slight but statistically significant improvement in 2,3-diphosphoglyceric acid concentration (p < 0.05). More notably, several markers of platelet damage were significantly and meaningfully higher in products generated with conventional centrifugation: the increase in platelet activation (assessed via P-selectin expression in platelets before and after blood processing) was nearly 4-fold higher for platelet units produced via centrifugation, and the release of pro-inflammatory mediators (soluble CD40-ligand, thromboxane B2) was significantly higher for centrifuged platelets as well (p < 0.01). CONCLUSION: This study demonstrated that a simple, passive system for separating donated blood into components may be a viable alternative to centrifugation-particularly for applications in remote or resource-limited settings, or for patients requiring highly functional platelet product.

Washing in hypotonic saline reduces the fraction of irreversibly-damaged cells in stored blood: a proof-of-concept study.

BACKGROUND: During hypothermic storage, a substantial fraction of red blood cells (RBCs) transforms from flexible discocytes to rigid sphero-echinocytes and spherocytes. Infusion of these irreversibly-damaged cells into the recipient during transfusion serves no therapeutic purpose and may contribute to adverse outcomes in some patients. In this proof-of-concept study we describe the use of hypotonic washing for selective removal of the irreversibly-damaged cells from stored blood. MATERIALS AND METHODS: Stored RBCs were mixed with saline of various concentrations to identify optimal concentration for inducing osmotic swelling and selective bursting of spherical cells (sphero-echinocytes, spherocytes), while minimising indiscriminate lysis of other RBCs. Effectiveness of optimal treatment was assessed by measuring morphology, rheological properties, and surface phosphatidylserine (PS) exposure for cells from several RBCs units (n=5, CPD>AS-1, leucoreduced, 6 weeks storage duration) washed in hypotonic vs isotonic saline. RESULTS: Washing in mildly hypotonic saline (0.585 g/dL, osmolality: 221.7±2.3 mmol/kg) reduced the fraction of spherical cells 3-fold from 9.5±3.4% to 3.2±2.8%, while cutting PS exposure in half from 1.48±0.86% to 0.59±0.29%. Isotonic washing had no effect on PS exposure or the fraction of spherical cells. Both isotonic and hypotonic washing increased the fraction of well-preserved cells (discocytes, echinocytes 1) substantially, and improved the ability of stored RBCs to perfuse an artificial microvascular network by approximately 25%, as compared with the initial sample. DISCUSSION: This study demonstrated that washing in hypotonic saline could selectively remove a significant fraction of the spherical and PS-exposing cells from stored blood, while significantly improving the rheological properties of remaining well-preserved RBCs. Further studies are needed to access the potential effect from hypotonic washing on transfusion outcomes.

A high-throughput microfluidic approach for 1000-fold leukocyte reduction of platelet-rich plasma.

Leukocyte reduction of donated blood products substantially reduces the risk of a number of transfusion-related complications. Current 'leukoreduction' filters operate by trapping leukocytes within specialized filtration material, while allowing desired blood components to pass through. However, the continuous release of inflammatory cytokines from the retained leukocytes, as well as the potential for platelet activation and clogging, are significant drawbacks of conventional 'dead end' filtration. To address these limitations, here we demonstrate our newly-developed 'controlled incremental filtration' (CIF) approach to perform high-throughput microfluidic removal of leukocytes from platelet-rich plasma (PRP) in a continuous flow regime. Leukocytes are separated from platelets within the PRP by progressively syphoning clarified PRP away from the concentrated leukocyte flowstream. Filtrate PRP collected from an optimally-designed CIF device typically showed a ~1000-fold (i.e. 99.9%) reduction in leukocyte concentration, while recovering >80% of the original platelets, at volumetric throughputs of ~1 mL/min. These results suggest that the CIF approach will enable users in many fields to now apply the advantages of microfluidic devices to particle separation, even for applications requiring macroscale flowrates.

Controlled incremental filtration: a simplified approach to design and fabrication of high-throughput microfluidic devices for selective enrichment of particles.

The number of microfluidic strategies aimed at separating particles or cells of a specific size within a continuous flow system continues to grow. The wide array of biomedical and other applications that would benefit from successful development of such technology has motivated the extensive research in this area over the past 15 years. However, despite promising advancements in microfabrication capabilities, a versatile approach that is suitable for a large range of particle sizes and high levels of enrichment, with a volumetric throughput sufficient for large-scale applications, has yet to emerge. Here we describe a straightforward method that enables the rapid design of microfluidic devices that are capable of enriching/removing particles within a complex aqueous mixture, with an unprecedented range of potential cutoff diameter (below 1 µm to above 100 µm) and an easily scalable degree of enrichment/filtration (up to 10-fold and well beyond). A simplified model of a new approach to crossflow filtration - controlled incremental filtration - was developed and validated for its ability to generate microfluidic devices that efficiently separate particles on the order of 1-10 µm, with throughputs of tens of µL min(-1), without the use of a pump. Precise control of the amount of fluid incrementally diverted at each filtration "gap" of the device allows for the gap size (~20 µm) to be much larger than the particles of interest, while the simplicity of the model allows for many thousands of these filtration points to be readily incorporated into a desired device design. This new approach should enable truly high-throughput microfluidic particle-separation devices to be generated, even by users only minimally experienced in fluid mechanics and microfabrication techniques.

The effects of additive solution pH and metabolic rejuvenation on anaerobic storage of red cells.

BACKGROUND: Red cell (RBC) storage can be extended to 9 weeks under anaerobic or alkaline conditions. Simultaneous use of these approaches has not provided additive benefit. Our objective was to determine whether anaerobic storage with acidified additive solution (AS) coupled with metabolic rejuvenation might further improve the benefits of anaerobic storage. STUDY DESIGN AND METHODS: RBC storage in AS with a pH value of 6.5, 7.4, or 8.3 in aerobic or anaerobic conditions was examined using a panel of in vitro biochemical and RBC markers. RBC rejuvenation during cold storage was also evaluated. A randomized crossover radiolabeled recovery study (eight subjects) evaluated anaerobic RBC storage using AS65 with cold rejuvenation for up to 16 weeks of storage. RESULTS: Adenosine triphosphate (ATP) and diphosphoglycerate acid (DPG) were better maintained in anaerobic storage than in aerobic storage. Acidic or neutral AS preserved ATP concentration better, while a neutral or basic pH AS favored maintenance of DPG levels at higher levels for a longer period. AS pH had less of an effect on exposure of phosphatidylserine (PS), vesicle protein release, and hemolysis. Rejuvenation of RBCs during cold, anaerobic storage resulted in increases in ATP and DPG levels and a reversal of PS exposure. Anaerobic storage of RBCs in pH 6.5 AS rejuvenated at 7 weeks of storage yielded RBC 24-hour recoveries of 77.3 +/- 12.5 percent after 10 weeks' storage time. After a second rejuvenation at Week 11, six subjects' units demonstrated a recovery of 75.9 +/- 7.3 percent at 12 weeks of storage. CONCLUSION: Extended RBC storage may be achieved using anaerobic conditions combined with low-pH AS and rejuvenation during storage.

A detailed study of time-dependent changes in human red blood cells: from reticulocyte maturation to erythrocyte senescence.

The use of microfabrication technology in the study of biological systems continues to grow rapidly in both prevalence and ascendancy. Customised microdevices that provide superior results than traditional macroscopic methods can be designed in order to investigate specific cell types and cellular processes. This study showed the benefit of this approach in precisely characterising the progressive losses of surface area and haemoglobin (Hb) content by the human red blood cell (RBC), from newborn reticulocyte to senescent erythrocyte. The high-throughput, multiparametric measurements made on individual cells with a specialised microdevice enabled, for the first time, delineation and quantification of the losses that occur during the two stages of the human RBC lifespan. Data acquired on tens of thousands of red cells showed that nearly as much membrane area is lost during the 1-2 d of reticulocyte maturation (c. 10-14%) as in the subsequent 4 months of erythrocyte ageing (c. 16-17%). The total decrease in Hb over the red cell lifespan is also estimated (c. 15%) and a model describing the complete time-course of diminishing mean RBC area and Hb is proposed. The relationship between the losses of Hb and area, and their possible influence on red cell lifespan, are discussed.

Direct measurement of the impact of impaired erythrocyte deformability on microvascular network perfusion in a microfluidic device.

The ability of red blood cells (RBCs, erythrocytes) to deform and pass through capillaries is essential for continual flow of blood in the microvasculature, which ensures an adequate supply of oxygen and nutrients, prompt removal of metabolic waste products, transport of drugs and hormones, and traffic of circulating cells to and from all living tissues. This paper presents a novel tool for evaluating the impact of impaired deformability of RBCs on the flow of blood in the microvasculature by directly measuring perfusion of a test microchannel network with dimensions and topology similar to the real microcirculation. The measurement of microchannel network perfusion is compared with RBC filtration -- a conventional assay of RBC deformability. In contrast to RBC filterability, network perfusion depends linearly on RBC deformability modulated by graded exposure to glutaraldehyde, showing a higher sensitivity to small changes of deformability. The direct measurement of microchannel network perfusion represents a new concept for the field of blood rheology and should prove beneficial for basic science and clinical applications.

A high-resolution, double-labeling method for the study of in vivo red blood cell aging.

BACKGROUND: Red blood cell (RBC) senescence is a process that has received considerable study, yet remains poorly understood. This has been primarily due to the difficulty in isolating a RBC cohort of narrowly distributed, well-defined age. Biotin labeling has previously been used to produce an identifiable cell cohort of known mean age; however, the variability of RBC age within the cohort is relatively large for most of its existence. Treatments typically employed on animal subjects to reduce this variability can perturb erythropoiesis and result in abnormal RBC aging. STUDY DESIGN AND METHODS: The objective of this study was to improve on the traditional in vivo biotinylation method by introducing a chemically distinct, second labeling step. In this case, digoxigenin was used to label cells 1 to 2 days before the injection of biotin. RESULTS: It was shown, in the rat, that two identifiable subpopulations of labeled RBCs can be followed over time: a broad, double-labeled cohort and a narrow, single-labeled cohort, the latter consisting of only those cells created between the first and second labeling steps. The utility of this technique was demonstrated by observing the age-dependent exposure of phosphatidylserine in the single-labeled RBCs. CONCLUSION: Its capacity to generate a cohort of narrowly distributed age, without the adverse effects associated with animal treatment, should make this a useful method for the study of RBC senescence.

Prototype of an in vitro model of the microcirculation.

We have used microfabrication technology to construct a network of microchannels, patterned after the dimensions and architecture of the mammalian microcirculation. The network is cast in transparent silicone elastomer and the channels are coated with silanated mPEG to provide lubrication. Flow of red and white blood cells through the network is readily visualized by the use of high-speed digital image acquisition. The acquired sequences of high-quality images are used to calculate hematocrits and rates of red cell movement in the microchannels. Our prototype system has significant advantages over scaled-up room-size experimental systems in that it permits experimentation with actual human blood cells. Experiments can be carried out under well-controlled conditions in a network of microchannels with precisely known dimensions using cell suspensions of defined composition. Moreover, there is no need to counteract or anticipate the host's adaptive responses that may confound live animal experiments. Notwithstanding its limitations, the current prototype demonstrates certain features characteristic of the microcirculation, such as parachute and bullet shapes of red cells deformed in capillary channels, rouleaux formation, plasma skimming, and the utilization of collateral flow pathways due to flow obstruction caused by a white cell blocking a microchannel. We present this device as a prototype scale-to-scale model of the mammalian microcirculation. Limitations of the system as well as a variety of possible applications are described.

Parallel microchannel-based measurements of individual erythrocyte areas and volumes.

We describe a microchannel device which utilizes a novel approach to obtain area and volume measurements on many individual red blood cells. Red cells are aspirated into the microchannels much as a single red blood cell is aspirated into a micropipette. Inasmuch as there are thousands of identical microchannels with defined geometry, data for many individual red cells can be rapidly acquired, and the fundamental heterogeneity of cell membrane biophysics can be analyzed. Fluorescent labels can be used to quantify red cell surface and cytosolic features of interest simultaneously with the measurement of area and volume for a given cell. Experiments that demonstrate and evaluate the microchannel measuring capabilities are presented and potential improvements and extensions are discussed.