Improved expansion of T cells in culture when isolated with an equipment-free, high-throughput, flow-through microfluidic module versus traditional density gradient centrifugation
Abstract: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 preciselydesigned, but… Show more
“…Because the flow fraction extracted through each gap is relatively small, any CIF design must incorporate thousands of gaps to achieve the desired filtrate:retentate flow ratio 43 , 44 . In practice, the number of filtration gaps (and, hence, the flow ratio) is limited by the maximum channel length allowed by the fabrication technique 44 , 46 . Figure 1 The separation of WBCs from RBCs and PLTs via controlled incremental filtration (CIF).…”
Section: Resultsmentioning
confidence: 99%
“…‘Controlled incremental filtration’ (CIF) overcame these limitations by having a substantially larger minimal feature size (~ 20 µm), and thus reducing fluidic resistance and shear throughout the device 43 , 44 . As a result, a CIF-based device was able to separate > 85% of WBCs (with < 30% loss of RBCs and PLTs) from the mononuclear cell (MNC) concentrates (~ 5% HCT) at flow rates of up to 30 mL/min 46 and PLTs concentrated and leukoreduced using CIF were minimally activated 43 , 44 . Notwithstanding these significant advancements, none of the aforementioned devices have been tested in the recirculation regime for any significant amount of time, and the effects of such processing on blood cells and device performance remain largely unknown.…”
Leukapheresis, the extracorporeal separation of white blood cells (WBCs) from red blood cells (RBCs) and platelets (PLTs), is a life-saving procedure used for treating patients with cancer and other conditions, and as the initial step in the manufacturing of cellular and gene-based therapies. Well-tolerated by adults, leukapheresis poses a significant risk to neonates and low-weight infants because the extracorporeal volume (ECV) of standard centrifugation-based machines represents a particularly large fraction of these patients’ total blood volume. Here we describe a novel high-throughput microfluidic device (with a void volume of 0.4 mL) based on controlled incremental filtration (CIF) technology that could replace centrifugation for performing leukapheresis. The CIF device was tested extensively using whole blood from healthy volunteers at multiple hematocrits (5–30%) and flow rates (10–30 mL/min). In the flow-through regime, the CIF device separated WBCs with > 85% efficiency and 10–15% loss of RBCs and PLTs while processing whole blood diluted with saline to 10% hematocrit at a flow rate of 10 mL/min. In the recirculation regime, the CIF device demonstrated a similar level of separation performance, virtually depleting WBCs in the recirculating blood (~ 98% reduction) by the end of a 3.5-hour simulated leukapheresis procedure. Importantly, the device operated without clogging or decline in separation performance, with minimal activation of WBCs and PLTs and no measurable damage to RBCs. Compared to the typical parameters of centrifugation-based leukapheresis, the CIF device had a void volume at least 100-fold smaller, removed WBCs about twice as fast, and lost ~ 2–3-fold fewer PLTs, while operating at a flow rate compatible with the current practice. The hematocrit and flow rate at which the CIF device operated were significantly higher than previously published for other microfluidic cell separation methods. Finally, this study is the first to demonstrate a highly efficient separation of cells from recirculating blood using a microfluidic device. Overall, these findings suggest the feasibility of using high-throughput microfluidic cell separation technology to ultimately enable centrifugation-free, low-ECV leukapheresis. Such a capability would be particularly useful in young children, a vulnerable group of patients who are currently underserved.
“…Because the flow fraction extracted through each gap is relatively small, any CIF design must incorporate thousands of gaps to achieve the desired filtrate:retentate flow ratio 43 , 44 . In practice, the number of filtration gaps (and, hence, the flow ratio) is limited by the maximum channel length allowed by the fabrication technique 44 , 46 . Figure 1 The separation of WBCs from RBCs and PLTs via controlled incremental filtration (CIF).…”
Section: Resultsmentioning
confidence: 99%
“…‘Controlled incremental filtration’ (CIF) overcame these limitations by having a substantially larger minimal feature size (~ 20 µm), and thus reducing fluidic resistance and shear throughout the device 43 , 44 . As a result, a CIF-based device was able to separate > 85% of WBCs (with < 30% loss of RBCs and PLTs) from the mononuclear cell (MNC) concentrates (~ 5% HCT) at flow rates of up to 30 mL/min 46 and PLTs concentrated and leukoreduced using CIF were minimally activated 43 , 44 . Notwithstanding these significant advancements, none of the aforementioned devices have been tested in the recirculation regime for any significant amount of time, and the effects of such processing on blood cells and device performance remain largely unknown.…”
Leukapheresis, the extracorporeal separation of white blood cells (WBCs) from red blood cells (RBCs) and platelets (PLTs), is a life-saving procedure used for treating patients with cancer and other conditions, and as the initial step in the manufacturing of cellular and gene-based therapies. Well-tolerated by adults, leukapheresis poses a significant risk to neonates and low-weight infants because the extracorporeal volume (ECV) of standard centrifugation-based machines represents a particularly large fraction of these patients’ total blood volume. Here we describe a novel high-throughput microfluidic device (with a void volume of 0.4 mL) based on controlled incremental filtration (CIF) technology that could replace centrifugation for performing leukapheresis. The CIF device was tested extensively using whole blood from healthy volunteers at multiple hematocrits (5–30%) and flow rates (10–30 mL/min). In the flow-through regime, the CIF device separated WBCs with > 85% efficiency and 10–15% loss of RBCs and PLTs while processing whole blood diluted with saline to 10% hematocrit at a flow rate of 10 mL/min. In the recirculation regime, the CIF device demonstrated a similar level of separation performance, virtually depleting WBCs in the recirculating blood (~ 98% reduction) by the end of a 3.5-hour simulated leukapheresis procedure. Importantly, the device operated without clogging or decline in separation performance, with minimal activation of WBCs and PLTs and no measurable damage to RBCs. Compared to the typical parameters of centrifugation-based leukapheresis, the CIF device had a void volume at least 100-fold smaller, removed WBCs about twice as fast, and lost ~ 2–3-fold fewer PLTs, while operating at a flow rate compatible with the current practice. The hematocrit and flow rate at which the CIF device operated were significantly higher than previously published for other microfluidic cell separation methods. Finally, this study is the first to demonstrate a highly efficient separation of cells from recirculating blood using a microfluidic device. Overall, these findings suggest the feasibility of using high-throughput microfluidic cell separation technology to ultimately enable centrifugation-free, low-ECV leukapheresis. Such a capability would be particularly useful in young children, a vulnerable group of patients who are currently underserved.
“…Reprinted from Cytotherapy , Vol. 21 , Strachan, B. C., Xia, H. U. I., Vörös, E., Gifford, S. C., Shevkoplyas, S. S., Improved expansion of T cells in culture when isolated with an equipment-free, high-throughput, flow-through microfluidic module versus traditional density gradient centrifugation, pp 234–245 (ref ). Copyright 2019, with permission from Elsevier.…”
Section: Sorting Methodsmentioning
confidence: 99%
“…Strachan et al developed a microfluidic device for the isolation of leukocytes based on a controlled incremental filtration (CIF) device that can operate with gravity to drive flow (Figure C–E). Blood cells that have an effective size greater than the threshold critical diameter of the device were retained in its central channel, whereas cells less than the cutoff size were able to be pulled into the side (i.e., filtrate channels) by the flow of fluid out of the central channel at each filtration point.…”
The development of new methods and technologies for cell separation, sorting, selection, isolation, or enrichment (many times the aforementioned terms are used interchangeably) are becoming more imperative due in part to the wealth of research aimed at addressing issues for the analysis of liquid biopsy targets, such as rare cells, to realize the concept of "precision medicine". Precision medicine is defined as "treatments targeted to the needs of individual patients on the basis of genetic, biomarker, phenotypic, or psychosocial characteristics that distinguish a patient from others with similar clinical presentations." 1 Before full implementation of precision medicine in a hospital or clinical setting, technological discoveries must be realized to assist in the analysis of disease-associated cells enriched from complex samples.Liquid biopsy markers and the cargo that they carry have been shown to be an attractive source of information that can be used to facilitate precise decisions for disease management. These biomarkers include, but are not limited to, rare cells such as circulating tumor cells (CTCs) or cancer stem cells (CSCs), immune cells (i.e., CD8+ T-cells), bacterial and viral cells, cell-free molecules such as cell free DNA (cfDNA), and extracellular vesicles (EVs). Liquid biopsies are attractive because of the minimally invasive nature of the sampling method (i.e., collection of peripheral blood, urine, saliva) to secure relevant biomarkers. These biomarkers can enable precision medicine decisions on managing a variety of diseases, including oncology and nononcology-related diseases. 2,3 Liquid biopsies *
“…Thus, this method cannot be used to evaluate the adhesion strength of a large number of cells but can provide a good thinking of cell viability evaluation based on shear stress applications. Nevertheless, the high-throughput screening of strong cell adhesion can be realized in microfluidic chips for their even forces exerted by a constant flow rate. − In this paper, cell viability is evaluated by detecting the heterogeneity of cell populations whose detection occasionality becomes larger as the reduction of the detection upper limit of cell populations. It means that the lower the detection upper limit of cell population, the lower the accuracy of cell viability assessment is.…”
Due to the heterogeneity of cancer cell populations, the traditional evaluation approach of cell viability based on the cell counting assay is quite inaccurate for the dose−response test of anticancer drugs, cell toxicology assays, and other biochemical stimulations. In this paper, an evaluation approach of cell viability based on the cell detachment assay in a single-channel integrated microfluidic chip is proposed to improve the accuracy of cell viability assessment. The electrodes are coated by fibronectin for specific cell adhesion, and it is biologically significant to study the cell detachment assay in vitro. The maximum number of cells that can be detected by this sensor is about 10 5 cells (overgrowing), while the minimum is about 100 cells. This method is calibrated with the half-maximal inhibitory concentration assay, and the results show that the cell viability calculated by adhesion strength is more accurate than that evaluated using the cell counting assay. Meanwhile, the shear rate is transformed into shear stress for the comparability among the results in other papers. The most sensitive frequency is also determined as 1 kHz according to normalized impedance. Besides, the impedance of cell adhesion affected by different shear stresses is monitored to study the optimized plan for long-term culture of cells in the integrated microfluidic chip prepared for the cell detachment assay. Adhesion strength τ 25 , which is the magnitude of shear stress needed to detach 75% of cell population, is introduced to describe the cell adhesion forces. It is calculated and normalized based on the cell detachment assay to evaluate cell viability. The relative errors of the cell detachment method compared with those of the cell counting method decrease by 0.637 (0% FBS), 0.586 (0.5% FBS), and 0.342 (2% FBS).
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