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1.
Methods Mol Biol ; 2804: 91-100, 2024.
Article En | MEDLINE | ID: mdl-38753142

Circulating tumor cells (CTCs) isolated directly from whole blood opens new perspectives for cancer monitoring and the development of personalized treatments. However, due to their rarity among the multitude of blood cells, it remains a challenge to recover them alive with high level of purity, i.e., with few remaining white blood cells, and in a time frame compatible with the clinical context. Microfluidic chips have emerged as promising tools to address these challenges. We propose a two-step workflow including a pre-enrichment step, performed by a size-based pre-enrichment system, and a purification step, performed by an immunomagnetic chip. Here, we describe the protocol for the fabrication of the immunomagnetic microchip, the preparation of the sample, and the procedure for injection into the microchip allowing the sorting of the CTCs.


Immunomagnetic Separation , Lab-On-A-Chip Devices , Neoplastic Cells, Circulating , Neoplastic Cells, Circulating/pathology , Immunomagnetic Separation/methods , Humans , Cell Separation/methods , Cell Separation/instrumentation , Neoplasms/pathology , Neoplasms/blood , Cell Line, Tumor , Microfluidic Analytical Techniques/instrumentation , Microfluidic Analytical Techniques/methods
2.
Reprod Domest Anim ; 59(5): e14583, 2024 May.
Article En | MEDLINE | ID: mdl-38747479

Testosterone, an important sex hormone, regulates sexual maturation, testicular development, spermatogenesis and the maintenance of secondary sexual characteristics in males. Testicular Leydig cells are the primary source of testosterone production in the body. Hezuo pigs, native to the southern part of Gansu, China, are characterized by early sexual maturity, strong disease resistance and roughage tolerance. This study employed type IV collagenase digestion combined with cell sieve filtration to isolate and purify Leydig cells from the testicular tissue of 1-month-old Hezuo pigs. We also preliminarily investigated the functions of these cells. The results indicated that the purity of the isolated and purified Leydig cells was as high as 95%. Immunofluorescence analysis demonstrated that the isolated cells specifically expressed the 3ß-hydroxysteroid dehydrogenase antibody. Enzyme-linked immunosorbent assay results showed that the testosterone secretion of the Leydig cells cultured in vitro (generations 5-9) ranged between 1.29-1.67 ng/mL. Additionally, the content of the cellular autophagy signature protein microtubule-associated protein 1 light chain 3 was measured at 230-280 pg/mL. Through this study, we established an in vitro system for the isolation, purification and characterization of testicular Leydig cells from 1-month-old Hezuo pigs, providing a reference for exploring the molecular mechanism behind precocious puberty in Hezuo pigs.


Leydig Cells , Testosterone , Animals , Male , Leydig Cells/metabolism , Testosterone/metabolism , Swine , Testis/cytology , Cells, Cultured , Cell Culture Techniques/veterinary , Cell Separation/methods , Cell Separation/veterinary
3.
Physiol Rep ; 12(9): e16040, 2024 May.
Article En | MEDLINE | ID: mdl-38725080

The endocrine pancreas is composed of clusters of cell groups called pancreatic islets. These cells are responsible for the synthesis and secretion of hormones crucial for glycemic homeostasis, such as insulin and glucagon. Therefore, these cells were the targets of many studies. One method to study and/or understand endocrine pancreatic physiology is the isolation of these islets and stimulation of hormone production using different concentrations of glucose, agonists, and/or antagonists of specific secretagogues and mimicking the stimulation of hormonal synthesis and secretion. Many researchers studied pancreatic physiology in murine models due to their ease of maintenance and rapid development. However, the isolation of pancreatic islets involves meticulous processes that may vary between rodent species. The present study describes a simple and effective technical protocol for isolating intact islets from mice and rats for use as a practical guide for researchers. The method involves digestion of the acinar parenchyma by intraductal collagenase. Isolated islets are suitable for in vitro endocrine secretion analyses, microscopy techniques, and biochemical analyses.


Islets of Langerhans , Animals , Islets of Langerhans/metabolism , Islets of Langerhans/cytology , Mice , Rats , Male , Mice, Inbred C57BL , Cell Separation/methods
4.
Methods Mol Biol ; 2800: 1-10, 2024.
Article En | MEDLINE | ID: mdl-38709473

The fruit fly Drosophila is a well-established invertebrate model that enables in vivo imaging of innate immune cell (e.g., macrophage) migration and signaling at high spatiotemporal resolution within the intact, living animal. While optimized methods already exist to enable flow cytometry-based macrophage isolation from Drosophila at various stages of development, there remains a need for more rapid and gentle methods to isolate living macrophages for downstream ex vivo applications. Here, we describe techniques for rapid and direct isolation of living macrophages from mature Drosophila pupae and their downstream ex vivo preparation for live imaging and immunostaining. This strategy enables straightforward access to physiologically relevant innate immune cells, both circulating and tissue-resident populations, for subsequent imaging of signal transduction.


Macrophages , Pupa , Animals , Pupa/cytology , Macrophages/cytology , Macrophages/metabolism , Drosophila , Cell Separation/methods , Flow Cytometry/methods , Drosophila melanogaster/cytology
5.
Stem Cell Res Ther ; 15(1): 131, 2024 May 03.
Article En | MEDLINE | ID: mdl-38702793

BACKGROUND: Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) hold great therapeutic potential in regenerative medicine. Therefore, it is crucial to establish a Good Manufacturing Practice (GMP)-compliant methodology for the isolation and culture of WJ-MSCs. Through comprehensive research, encompassing laboratory-scale experiments to pilot-scale studies, we aimed to develop standardized protocols ensuring the high yield and quality of WJ-MSCs manufacturing. METHODS: Firstly, optimization of parameters for the enzymatic digestion method used to isolate WJ-MSCs was conducted. These parameters included enzyme concentrations, digestion times, seeding densities, and culture media. Additionally, a comparative analysis between the explant method and the enzymatic digestion method was performed. Subsequently, the consecutive passaging of WJ-MSCs, specifically up to passage 9, was evaluated using the optimized method. Finally, manufacturing processes were developed and scaled up, starting from laboratory-scale flask-based production and progressing to pilot-scale cell factory-based production. Furthermore, a stability study was carried out to assess the storage and use of drug products (DPs). RESULTS: The optimal parameters for the enzymatic digestion method were a concentration of 0.4 PZ U/mL Collagenase NB6 and a digestion time of 3 h, resulting in a higher yield of P0 WJ-MSCs. In addition, a positive correlation between the weight of umbilical cord tissue and the quantities of P0 WJ-MSCs has been observed. Evaluation of different concentrations of human platelet lysate revealed that 2% and 5% concentrations resulted in similar levels of cell expansion. Comparative analysis revealed that the enzymatic digestion method exhibited faster outgrowth of WJ-MSCs compared to the explant method during the initial passage. Passages 2 to 5 exhibited higher viability and proliferation ability throughout consecutive passaging. Moreover, scalable manufacturing processes from the laboratory scale to the pilot scale were successfully developed, ensuring the production of high-quality WJ-MSCs. Multiple freeze-thaw cycles of the DPs led to reduced cell viability and viable cell concentration. Subsequent thawing and dilution of the DPs resulted in a significant decrease in both metrics, especially when stored at 20-27 °C. CONCLUSION: This study offers valuable insights into optimizing the isolation and culture of WJ-MSCs. Our scalable manufacturing processes facilitate the large-scale production of high-quality WJ-MSCs. These findings contribute to the advancement of WJ-MSCs-based therapies in regenerative medicine.


Mesenchymal Stem Cells , Wharton Jelly , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Humans , Wharton Jelly/cytology , Cell Culture Techniques/methods , Cell Differentiation , Cells, Cultured , Cell Proliferation , Cell Separation/methods , Cell Separation/standards
6.
J Transl Med ; 22(1): 459, 2024 May 15.
Article En | MEDLINE | ID: mdl-38750573

Mesenchymal stem/stromal cells (MSCs) represent a heterogeneous cell population distributed throughout various tissues, demonstrating remarkable adaptability to microenvironmental cues and holding immense promise for disease treatment. However, the inherent diversity within MSCs often leads to variability in therapeutic outcomes, posing challenges for clinical applications. To address this heterogeneity, purification of MSC subpopulations through marker-based isolation has emerged as a promising approach to ensure consistent therapeutic efficacy. In this review, we discussed the reported markers of MSCs, encompassing those developed through candidate marker strategies and high-throughput approaches, with the aim of explore viable strategies for addressing the heterogeneity of MSCs and illuminate prospective research directions in this field.


Biomarkers , Mesenchymal Stem Cells , Humans , Mesenchymal Stem Cells/cytology , Biomarkers/metabolism , Animals , Cell Separation/methods
7.
Biosensors (Basel) ; 14(5)2024 May 09.
Article En | MEDLINE | ID: mdl-38785711

Electrokinetic (EK) microsystems, which are capable of performing separations without the need for labeling analytes, are a rapidly growing area in microfluidics. The present work demonstrated three distinct binary microbial separations, computationally modeled and experimentally performed, in an insulator-based EK (iEK) system stimulated by DC-biased AC potentials. The separations had an increasing order of difficulty. First, a separation between cells of two distinct domains (Escherichia coli and Saccharomyces cerevisiae) was demonstrated. The second separation was for cells from the same domain but different species (Bacillus subtilis and Bacillus cereus). The last separation included cells from two closely related microbial strains of the same domain and the same species (two distinct S. cerevisiae strains). For each separation, a novel computational model, employing a continuous spatial and temporal function for predicting the particle velocity, was used to predict the retention time (tR,p) of each cell type, which aided the experimentation. All three cases resulted in separation resolution values Rs>1.5, indicating complete separation between the two cell species, with good reproducibility between the experimental repetitions (deviations < 6%) and good agreement (deviations < 18%) between the predicted tR,p and experimental (tR,e) retention time values. This study demonstrated the potential of DC-biased AC iEK systems for performing challenging microbial separations.


Saccharomyces cerevisiae , Escherichia coli , Lab-On-A-Chip Devices , Bacillus cereus , Microfluidic Analytical Techniques , Cell Separation/methods , Bacillus subtilis
8.
Mol Brain ; 17(1): 25, 2024 May 21.
Article En | MEDLINE | ID: mdl-38773624

A growing body of evidence indicates intra- and inter-regional heterogeneity of astrocytes in the brain. However, because of a lack of an efficient method for isolating astrocytes from the spinal cord, little is known about how much spinal cord astrocytes are heterogeneous in adult mice. In this study, we developed a new method for isolating spinal astrocytes from adult mice using a cold-active protease from Bacillus licheniformis with an astrocyte cell surface antigen-2 (ACSA-2) antibody. Using fluorescence-activated cell sorting, isolated spinal ACSA-2+ cells were divided into two distinct populations, ACSA-2high and ACSA-2low. By analyzing the expression of cell-type marker genes, the ACSA-2high and ACSA-2low populations were identified as astrocytes and ependymal cells, respectively. Furthermore, ACSA-2high cells had mRNAs encoding genes that were abundantly expressed in the gray matter (GM) but not white matter astrocytes. By optimizing enzymatic isolation procedures, the yield of GM astrocytes also increased. Therefore, our newly established method enabled the selective and efficient isolation of GM astrocytes from the spinal cord of adult mice and may be useful for bulk- or single-cell RNA-sequencing under physiological and pathological conditions.


Astrocytes , Cell Separation , Gray Matter , Spinal Cord , Animals , Astrocytes/metabolism , Astrocytes/cytology , Spinal Cord/cytology , Cell Separation/methods , Mice, Inbred C57BL , Mice , Male , RNA, Messenger/metabolism , RNA, Messenger/genetics , Aging
9.
Front Immunol ; 15: 1368118, 2024.
Article En | MEDLINE | ID: mdl-38756770

Frequencies and phenotypes of immune cells differ between neonates and adults in association with age-specific immune responses. Lymph nodes (LN) are critical tissue sites to quantify and define these differences. Advances in flow cytometry have enabled more multifaceted measurements of complex immune responses. Tissue processing can affect the immune cells under investigation that influence key findings. To understand the impact on immune cells in the LN after processing for single-cell suspension, we compared three dissociation protocols: enzymatic digestion, mechanical dissociation with DNase I treatment, and mechanical dissociation with density gradient separation. We analyzed cell yields, viability, phenotypic and maturation markers of immune cells from the lung-draining LN of neonatal and adult mice two days after intranasal respiratory syncytial virus (RSV) infection. While viability was consistent across age groups, the protocols influenced the yield of subsets defined by important phenotypic and activation markers. Moreover, enzymatic digestion did not show higher overall yields of conventional dendritic cells and macrophages from the LN. Together, our findings show that the three dissociation protocols have similar impacts on the number and viability of cells isolated from the neonatal and adult LN. However, enzymatic digestion impacts the mean fluorescence intensity of key lineage and activation markers that may influence experimental findings.


Animals, Newborn , Lymph Nodes , Lymphocytes , Myeloid Cells , Phenotype , Respiratory Syncytial Virus Infections , Animals , Lymph Nodes/immunology , Lymph Nodes/cytology , Mice , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus Infections/virology , Lymphocytes/immunology , Lymphocytes/metabolism , Myeloid Cells/immunology , Cell Separation/methods , Flow Cytometry/methods , Immunophenotyping , Female , Mice, Inbred C57BL , Dendritic Cells/immunology , Dendritic Cells/metabolism
10.
Biosens Bioelectron ; 258: 116353, 2024 Aug 15.
Article En | MEDLINE | ID: mdl-38696966

Male infertility is a pervasive global reproductive challenge, primarily attributed to a decline in semen quality. Addressing this concern, there has been a growing focus on spermatozoa sorting in assisted reproductive technology. This study introduces a groundbreaking development in the form of a thermotaxis and rheotaxis microfluidic (TRMC) device designed for efficient motile spermatozoa sorting within a short 15-min timeframe. The TRMC device mimics the natural sperm sorting mechanism of the oviduct, selecting spermatozoa with superior motility and DNA integrity. The experimental outcomes demonstrate a remarkable enhancement in the percentage of progressive spermatozoa following sorting, soaring from 3.90% to an impressive 96.11% when subjected to a temperature decrease from 38 °C to 35 °C. Notably, sperm motility exhibited a substantial 69% improvement. The TRMC device exhibited a commendable recovery rate of 60.93%, surpassing current clinical requirements. Furthermore, the sorted spermatozoa displayed a notable reduction in the DNA fragmentation index to 6.94%, signifying a substantial 90% enhancement in DNA integrity. This remarkable advancement positions the TRMC device as highly suitable for applications in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), offering a promising solution to male infertility challenges.


Lab-On-A-Chip Devices , Sperm Motility , Spermatozoa , Male , Spermatozoa/physiology , Spermatozoa/cytology , Humans , Equipment Design , Infertility, Male , Biosensing Techniques/instrumentation , Cell Separation/instrumentation , DNA Fragmentation , Temperature
11.
Mikrochim Acta ; 191(5): 295, 2024 05 03.
Article En | MEDLINE | ID: mdl-38700804

White blood cells (WBCs) are robust defenders during antigenic challenges and prime immune cell functioning indicators. High-purity WBC separation is vital for various clinical assays and disease diagnosis. Red blood cells (RBCs) are a major hindrance in WBC separation, constituting 1000 times the WBC population. The study showcases a low-cost micropump integrated microfluidic platform to provide highly purified WBCs for point-of-care testing. An integrated user-friendly microfluidic platform was designed to separate WBCs from finger-prick blood (⁓5 µL), employing an inertial focusing technique. We achieved an efficient WBC separation with 86% WBC purity and 99.99% RBC removal rate in less than 1 min. In addition, the microdevice allows lab-on-chip colorimetric evaluation of chronic granulomatous disease (CGD), a rare genetic disorder affecting globally. The assay duration, straight from separation to disease detection, requires only 20 min. Hence, the proposed microfluidic platform can further be implemented to streamline various clinical procedures involving WBCs in healthcare industries.


Cell Separation , Granulomatous Disease, Chronic , Lab-On-A-Chip Devices , Leukocytes , Microfluidic Analytical Techniques , Humans , Granulomatous Disease, Chronic/diagnosis , Granulomatous Disease, Chronic/blood , Leukocytes/cytology , Cell Separation/instrumentation , Cell Separation/methods , Microfluidic Analytical Techniques/instrumentation , Microfluidic Analytical Techniques/methods
12.
J Nanobiotechnology ; 22(1): 231, 2024 May 08.
Article En | MEDLINE | ID: mdl-38720360

BACKGROUND: Circulating tumor cells (CTCs) are considered as a useful biomarker for early cancer diagnosis, which play a crucial role in metastatic process. Unfortunately, the tumor heterogeneity and extremely rare occurrence rate of CTCs among billions of interfering leukocytes seriously hamper the sensitivity and purity of CTCs isolation. METHODS: To address these, we firstly used microfluidic chips to detect the broad-spectrum of triple target combination biomarkers in CTCs of 10 types of cancer patients, including EpCAM, EGFR and Her2. Then, we constructed hybrid engineered cell membrane-camouflaged magnetic nanoparticles (HE-CM-MNs) for efficient capture of heterogeneous CTCs with high-purity, which was enabled by inheriting the recognition ability of HE-CM for various CTCs and reducing homologous cell interaction with leukocytes. Compared with single E-CM-MNs, HE-CM-MNs showed a significant improvement in the capture efficiency for a cell mixture, with an efficiency of 90%. And the capture efficiency of HE-CM-MNs toward 12 subpopulations of tumor cells was ranged from 70 to 85%. Furthermore, by using HE-CM-MNs, we successfully isolated heterogeneous CTCs with high purity from clinical blood samples. Finally, the captured CTCs by HE-CM-MNs could be used for gene mutation analysis. CONCLUSIONS: This study demonstrated the promising potential of HE-CM-MNs for heterogeneous CTCs detection and downstream analysis.


Biomarkers, Tumor , Cell Membrane , Cell Separation , Magnetite Nanoparticles , Neoplastic Cells, Circulating , Neoplastic Cells, Circulating/pathology , Neoplastic Cells, Circulating/metabolism , Humans , Magnetite Nanoparticles/chemistry , Cell Separation/methods , Cell Line, Tumor , Cell Membrane/metabolism , Cell Membrane/chemistry , Biomarkers, Tumor/blood , Receptor, ErbB-2 , Epithelial Cell Adhesion Molecule/metabolism , ErbB Receptors/genetics , ErbB Receptors/metabolism , Neoplasms
13.
Int J Mol Sci ; 25(9)2024 Apr 30.
Article En | MEDLINE | ID: mdl-38732108

Platelets are metabolically active, anucleated and small circulating cells mainly responsible for the prevention of bleeding and maintenance of hemostasis. Previous studies showed that platelets mitochondrial content, function, and energy supply change during several diseases such as HIV/AIDS, COVID-19, pulmonary arterial hypertension, and in preeclampsia during pregnancy. These changes in platelets contributed to the severity of diseases and mortality. In our previous studies, we have shown that the seahorse-based cellular stress assay (CSA) parameters are crucial to the understanding of the mitochondrial performance in peripheral blood mononuclear cells (PBMCS). Moreover, the results of CSA parameters were significantly influenced by the PBMC preparation methods. In this study, we assessed the correlation of CSA parameters and intracellular ATP content in platelets and evaluated the effects of platelet preparation methods on the results of CSA parameters and intracellular ATP content. We compared the results of CSA parameters and intracellular ATP content in platelets isolated by density centrifugation with Optiprep and simple centrifugation of blood samples without Optiprep. Platelets isolated by centrifugation with Optiprep showed a higher spare capacity, basal respiration, and maximal respiration than those isolated without Optiprep. There was a clear correlation between basal respiration and maximal respiration, and the whole-ATP content in both isolation methods. Moreover, a positive correlation was observed between the relative spare capacity and whole-cell ATP content. In conclusion, the results of seahorse-based CSA parameters and intracellular ATP content in platelets are markedly influenced by the platelet isolation methods employed. The results of basal respiration and maximal respiration are hallmarks of cellular activity in platelets, and whole-cell ATP content is a potential hint for basic platelet viability. We recommend further studies to evaluate the role of CSA parameters and intracellular ATP content in platelets as biomarkers for the diagnosis and prediction of disease states.


Adenosine Triphosphate , Blood Platelets , Humans , Blood Platelets/metabolism , Adenosine Triphosphate/metabolism , Adult , Mitochondria/metabolism , Stress, Physiological , Female , Cell Separation/methods , Leukocytes, Mononuclear/metabolism , Male , Middle Aged
14.
Anim Reprod Sci ; 265: 107493, 2024 Jun.
Article En | MEDLINE | ID: mdl-38701639

Not all boar sperm samples survive cryopreservation well. A method of eliminating damaged sperm might enable more cryopreserved boar semen to be used for pig breeding. In this study we investigated the use of Magnetic Activated Cell sorting (MACS) to eliminate damaged sperm from thawed boar semen samples. The thawed samples were mixed with Dead cell removal particles and were applied to the column in a SuperMACS II. Different fractions were collected: Original sample (O), Flow-through (FT), and Eluate (E). Sperm membrane integrity, mitochondrial membrane potential and reactive oxygen species were evaluated by flow cytometry after staining with SYBR 14 and propidium iodide, or 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide, or hydroethidine and dichlorodihydrofluorescein diacetate, respectively. The FT samples had increased membrane integrity, a greater proportion of sperm with high mitochondrial membrane potential and a greater proportion of sperm negative for hydrogen peroxide than O samples (P<0.0001), which in turn had increased membrane integrity than E samples (P <0.0001). However, differences were seen between boars. The FT samples had increased values of live, superoxide positive sperm than O samples (P <0.0001) and O samples had greater values than E samples (P <0.0001), while there was no effect of boar. Sperm quality was best in the FT fraction, comprising approximately 32% of the sperm sample. In conclusion, although there were differences between boars, MACS separation can improve sperm quality in thawed semen samples. It would be interesting to see if this improvement is reflected in fertility outcomes.


Cryopreservation , Semen Preservation , Spermatozoa , Animals , Male , Spermatozoa/physiology , Swine/physiology , Semen Preservation/veterinary , Semen Preservation/methods , Cryopreservation/veterinary , Cryopreservation/methods , Cell Membrane/physiology , Membrane Potential, Mitochondrial/physiology , Cell Separation/veterinary , Cell Separation/methods , Flow Cytometry/veterinary , Reactive Oxygen Species/metabolism , Semen Analysis/veterinary
15.
Chem Commun (Camb) ; 60(41): 5427-5430, 2024 May 16.
Article En | MEDLINE | ID: mdl-38685869

Synthetic mRNA circuits commonly sense input to produce binary output signals for cell separation. Based on virus-origin cap-independent translation initiation machinery and RBP-aptamer interaction, we designed smart synthetic mRNA-based circuits that sense single input molecules to bidirectionally tune output signals in an orthogonal manner, enabling high-resolution separation of cell populations.


Aptamers, Nucleotide , Cell Separation , RNA, Messenger , RNA, Messenger/genetics , RNA, Messenger/metabolism , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/metabolism , Humans , Cell Separation/methods
16.
Anal Methods ; 16(15): 2368-2377, 2024 Apr 18.
Article En | MEDLINE | ID: mdl-38572530

Microfluidic technology has great advantages in the precise manipulation of micro-nano particles, and the hybrid microfluidic separation technology has attracted much attention due to the advantages of both active and passive separation technology at the same time. In this paper, the hydrophoresis sorting technique is combined with the dielectrophoresis technique, and a dielectrophoresis-assisted hydrophoresis microdevice is studied to separate blood cells. By using the dielectrophoresis force to change the suspension position of the cells in the channel, the scope of the hydrophoresis device for sorting particles is expanded. At the same time, the effects of microchannel width, fluid velocity, and electrode voltage on cell sorting were discussed, and the cell separation process was simulated. This work has laid a certain theoretical foundation for the rapid diagnosis of diseases in practical applications.


Microfluidic Analytical Techniques , Microfluidic Analytical Techniques/methods , Computer Simulation , Microfluidics , Electrophoresis/methods , Cell Separation/methods
17.
J Immunol Methods ; 528: 113667, 2024 May.
Article En | MEDLINE | ID: mdl-38574803

Chimeric antigen receptor (CAR) redirected T cells are successfully employed in the combat against several hematological malignancies, however, are often compromised by low transduction rates making refinement of the CAR T cell products necessary. Here, we report a broadly applicable enrichment protocol relying on marking CAR T cells with an anti-glycine4-serine (G4S) linker antibody followed by magnetic activated cell sorting (MACS). The protocol is broadly applicable since the G4S peptide is an integral part of the vast majority of CARs as it links the VH and VL recognition domains. We demonstrate the feasibility by using the canonical second generation CARs specific for CEA and Her2, respectively, obtaining highly purified CAR T cell products in a one-step procedure without impairing cell viability. The protocol is also applicable to a dual specific CAR (tandem CAR). Except for CD39, T cell activation/exhaustion markers were not upregulated after separation. Purified CAR T cells retained their functionality with respect to antigen-specific cytokine secretion, cytotoxicity, and the capacity to proliferate and eliminate cognate tumor cells upon repetitive stimulation. Collectively, the one-step protocol for purifying CAR T cells extends the toolbox for preclinical research and specifically for clinical CAR T cell manufacturing.


Receptors, Chimeric Antigen , T-Lymphocytes , Cytotoxicity, Immunologic , Cell Separation , Magnetic Phenomena , Immunotherapy, Adoptive/methods
18.
Anal Chem ; 96(18): 6898-6905, 2024 May 07.
Article En | MEDLINE | ID: mdl-38649796

High-throughput single-cell analysis typically relies on the isolation of cells of interest in separate compartments for subsequent phenotypic or genotypic characterization. Using microfluidics, this is achieved by isolating individual cells in microdroplets or microwells. However, due to cell-to-cell variability in size, shape, and density, the cell capture efficiencies may vary significantly. This variability can negatively impact the measurements and introduce undesirable artifacts when trying to isolate and characterize heterogeneous cell populations. In this study, we show that single-cell isolation biases in microfluidics can be circumvented by increasing the viscosity of fluids in which cells are dispersed. At a viscosity of 40-50 cP (cP), the cell sedimentation is effectively reduced, resulting in a steady cell flow inside the microfluidics chip and consistent encapsulation in water-in-oil droplets over extended periods of time. This approach allows nearly all cells in a sample to be isolated with the same efficiency, irrespective of their type. Our results show that increased fluid viscosity, rather than cell-adjusted density, provides a more reliable approach to mitigate single-cell isolation biases.


Single-Cell Analysis , Viscosity , Humans , Microfluidic Analytical Techniques , Cell Separation/methods
19.
Anal Chem ; 96(18): 6914-6921, 2024 May 07.
Article En | MEDLINE | ID: mdl-38655666

BACKGROUND: There are important unmet clinical needs to develop cell enrichment technologies to enable unbiased label-free isolation of both single cell and clusters of circulating tumor cells (CTCs) manifesting heterogeneous lineage specificity. Here, we report a pilot study based on the microfluidic acoustophoresis enrichment of CTCs using the CellSearch CTC assay as a reference modality. METHODS: Acoustophoresis uses an ultrasonic standing wave field to separate cells based on biomechanical properties (size, density, and compressibility), resulting in inherently label-free and epitope-independent cell enrichment. Following red blood cell lysis and paraformaldehyde fixation, 6 mL of whole blood from 12 patients with metastatic prostate cancer and 20 healthy controls were processed with acoustophoresis and subsequent image cytometry. RESULTS: Acoustophoresis enabled enrichment and characterization of phenotypic CTCs (EpCAM+, Cytokeratin+, DAPI+, CD45-/CD66b-) in all patients with metastatic prostate cancer and detected CTC-clusters composed of only CTCs or heterogeneous aggregates of CTCs clustered with various types of white blood cells in 9 out of 12 patients. By contrast, CellSearch did not detect any CTC clusters, but detected comparable numbers of phenotypic CTCs as acoustophoresis, with trends of finding a higher number of CTCs using acoustophoresis. CONCLUSION: Our preliminary data indicate that acoustophoresis provides excellent possibilities to detect and characterize CTC clusters as a putative marker of metastatic disease and outcomes. Moreover, acoustophoresis enables the sensitive label-free enrichment of cells with epithelial phenotypes in blood and offers opportunities to detect and characterize CTCs undergoing epithelial-to-mesenchymal transitioning and lineage plasticity.


Cell Separation , Neoplastic Cells, Circulating , Prostatic Neoplasms , Humans , Male , Neoplastic Cells, Circulating/pathology , Prostatic Neoplasms/pathology , Prostatic Neoplasms/blood , Cell Separation/methods , Acoustics , Pilot Projects , Neoplasm Metastasis , Microfluidic Analytical Techniques
20.
Sci Rep ; 14(1): 8748, 2024 04 16.
Article En | MEDLINE | ID: mdl-38627566

Efficient techniques for separating target cells from undiluted blood are necessary for various diagnostic and research applications. This paper presents acoustic focusing in dense media containing iodixanol to purify peripheral blood mononuclear cells (PBMCs) from whole blood in a label-free and flow-through format. If the blood is laminated or mixed with iodixanol solutions while passing through the resonant microchannel, all the components (fluids and cells) rearrange according to their acoustic impedances. Red blood cells (RBCs) have higher effective acoustic impedance than PBMCs. Therefore, they relocate to the pressure node despite the dense medium, while PBMCs stay near the channel walls due to their negative contrast factor relative to their surrounding medium. By modifying the medium and thus tuning the contrast factor of the cells, we enriched PBMCs relative to RBCs by a factor of 3600 to 11,000 and with a separation efficiency of 85%. That level of RBC depletion is higher than most other microfluidic methods and similar to that of density gradient centrifugation. The current acoustophoretic chip runs up to 20 µl/min undiluted whole blood and can be integrated with downstream analysis.


Leukocytes, Mononuclear , Microfluidic Analytical Techniques , Cell Separation/methods , Triiodobenzoic Acids , Acoustics , Microfluidic Analytical Techniques/methods
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