Fast and efficient isolation of murine circulating tumor cells using screencell technology for pre-clinical analyzes.

Circulating tumor cells (CTCs) represent a rare and heterogeneous population of cancer cells that are detached from the tumor site and entered blood or lymphatic circulation. Once disseminated in distant tissues, CTCs could remain dormant or create a tumor mass causing serious danger for patients. Many technologies exist to isolate CTCs from patients' blood samples, mostly based on microfluidic systems or by sorting them according to their surface antigens, notably EpCAM, and/or cytokeratins for carcinoma. ScreenCell has developed an easy-to-use, antigen-independent, rapid, cost-effective, and efficient technology that isolates CTCs according to their bigger size compared to the blood cells. This study provides the technical information necessary to isolate and characterize CTCs from mouse blood. By using blood samples from transgenic mice with breast cancer or from WT mice in which we spiked cancer cells, we showed that ScreenCell technology is compatible with standard EDTA blood collection tubes. Furthermore, the ScreenCell Cyto kit could treat up to 500 µl and the ScreenCell MB kit up to 200 µl of mouse blood. As the ScreenCell MB kit captures unaltered live CTCs, we have shown that their DNA could be efficiently extracted, and the isolated cells could be grown in culture. In conclusion, ScreenCell provides a rapid, easy, antigen-independent, cost-effective, and efficient technology to isolate and characterize CTCs from the blood samples of cancer patients and murine models. Thanks to this technology CTCs could be captured fixed or alive. Murine cancer models are extensively used in pre-clinical studies. Therefore, this study demonstrates the crucial technical points necessary while manipulating mouse blood samples using ScreenCell technology.

Design and Application of Microfluidic Capture Device for Physical-Magnetic Isolation of MCF-7 Circulating Tumor Cells.

Circulating tumor cells (CTCs) are a type of cancer cell that spreads from the main tumor to the bloodstream, and they are often the most important among the various entities that can be isolated from the blood. For the diagnosis of cancer, conventional biopsies are often invasive and unreliable, whereas a liquid biopsy, which isolates the affected item from blood or lymph fluid, is a less invasive and effective diagnostic technique. Microfluidic technologies offer a suitable channel for conducting liquid biopsies, and this technology is utilized to extract CTCs in a microfluidic chip by physical and bio-affinity-based techniques. This effort uses functionalized magnetic nanoparticles (MNPs) in a unique microfluidic chip to collect CTCs using a hybrid (physical and bio-affinity-based/guided magnetic) capturing approach with a high capture rate. Accordingly, folic acid-functionalized Fe3O4 nanoparticles have been used to capture MCF-7 (breast cancer) CTCs with capture efficiencies reaching up to 95% at a 10 µL/min flow rate. Moreover, studies have been conducted to support this claim, including simulation and biomimetic investigations.

Isolation and adipogenic differentiation of murine mesenchymal stem cells harvested from macrophage-depleted bone marrow and adipose tissue.

INTRODUCTION AND PURPOSE: Mouse mesenchymal stem cells (MSCs) provide a resourceful tool to study physiological and pathological aspects of adipogenesis. Bone marrow-derived MSCs (BM-MSCs) and adipose tissue-derived MSCs (ASCs) are widely used for these studies. Since there is a wide spectrum of methods available, the purpose is to provide a focused hands-on procedural guide for isolation and characterization of murine BM-MSCs and ASCs and to effectively differentiate them into adipocytes. METHODS AND RESULTS: Optimized harvesting procedures for murine BM-MSCs and ASCs are described and graphically documented. Since macrophages reside in bone-marrow and fat tissues and regulate the biological behaviour of BM-MSCs and ASCs, we included a procedure to deplete macrophages from the MSC preparations. The identity and stemness of BM-MSCs and ASCs were confirmed by flow cytometry using established markers. Since the composition and concentrations of adipogenic differentiation cocktails differ widely, we present a standardized four-component adipogenic cocktail, consisting of insulin, dexamethasone, 3-isobutyl-1-methylxanthine, and indomethacin to efficiently differentiate freshly isolated or frozen/thawed BM-MSCs and ASCs into adipocytes. We further included visualization and quantification protocols of the differentiated adipocytes. CONCLUSION: This laboratory protocol was designed as a step-by-step procedure for harvesting murine BM-MSCs and ASCs and differentiating them into adipocytes.

Optimizing cell therapy by sorting cells with high extracellular vesicle secretion.

Critical challenges remain in clinical translation of extracellular vesicle (EV)-based therapeutics due to the absence of methods to enrich cells with high EV secretion. Current cell sorting methods are limited to surface markers that are uncorrelated to EV secretion or therapeutic potential. Here, we utilize a nanovial technology for enrichment of millions of single cells based on EV secretion. This approach is applied to select mesenchymal stem cells (MSCs) with high EV secretion as therapeutic cells for improving treatment. The selected MSCs exhibit distinct transcriptional profiles associated with EV biogenesis and vascular regeneration and maintain high levels of EV secretion after sorting and regrowth. In a mouse model of myocardial infarction, treatment with high-secreting MSCs improves heart functions compared to treatment with low-secreting MSCs. These findings highlight the therapeutic importance of EV secretion in regenerative cell therapies and suggest that selecting cells based on EV secretion could enhance therapeutic efficacy.

Purification and analysis of kidney-infiltrating leukocytes in a mouse model of lupus nephritis.

Renal injury often occurs as a complication in autoimmune diseases such as systemic lupus erythematosus (SLE). It is estimated that a minimum of 20% SLE patients develop lupus nephritis, a condition that can be fatal when the pathology progresses to end-stage renal disease. Studies in animal models showed that incidence of immune cell infiltrates in the kidney was linked to pathological injury and correlated with severe lupus nephritis. Thus, preventing immune cell infiltration into the kidney is a potential approach to impede the progression to an end-stage disease. A requirement to investigate the role of kidney-infiltrating leukocytes is the development of reproducible and efficient protocols for purification and characterization of immune cells in kidney samples. This chapter describes a detailed methodology that discriminates tissue-resident leukocytes from blood-circulating cells that are found in kidney. Our protocol was designed to maximize cell viability and to reduce variability among samples, with a combination of intravascular staining and magnetic bead separation for leukocyte enrichment. Experiments included as example were performed with FcγRIIb[KO] mice, a well-characterized murine model of SLE. We identified T cells and macrophages as the primary leukocyte subsets infiltrating into the kidney during severe nephritis, and we extensively characterized them phenotypically by flow cytometry.

A High-Throughput Circular Tumor Cell Sorting Chip with Trapezoidal Cross Section.

Circulating tumor cells are typically found in the peripheral blood of patients, offering a crucial pathway for the early diagnosis and prediction of cancer. Traditional methods for early cancer diagnosis are inefficient and inaccurate, making it difficult to isolate tumor cells from a large number of cells. In this paper, a new spiral microfluidic chip with asymmetric cross-section is proposed for rapid, high-throughput, label-free enrichment of CTCs in peripheral blood. A mold of the desired flow channel structure was prepared and inverted to make a trapezoidal cross-section using a micro-nanotechnology process of 3D printing. After a systematic study of how flow rate, channel width, and particle concentration affect the performance of the device, we utilized the device to simulate cell sorting of 6 µm, 15 µm, and 25 µm PS (Polystyrene) particles, and the separation efficiency and separation purity of 25 µm PS particles reached 98.3% and 96.4%. On this basis, we realize the enrichment of a large number of CTCs in diluted whole blood (5 mL). The results show that the separation efficiency of A549 was 88.9% and the separation purity was 96.4% at a high throughput of 1400 µL/min. In conclusion, we believe that the developed method is relevant for efficient recovery from whole blood and beneficial for future automated clinical analysis.

Biomechanics of circulating cellular and subcellular bioparticles: beyond separation.

Biomechanical attributes have emerged as novel markers, providing a reliable means to characterize cellular and subcellular fractions. Numerous studies have identified correlations between these factors and patients' medical status. However, the absence of a thorough overview impedes their applicability in contemporary state-of-the-art therapeutic strategies. In this context, we provide a comprehensive analysis of the dimensions, configuration, rigidity, density, and electrical characteristics of normal and abnormal circulating cells. Subsequently, the discussion broadens to encompass subcellular bioparticles, such as extracellular vesicles (EVs) enriched either from blood cells or other tissues. Notably, cell sizes vary significantly, from 2 µm for platelets to 25 µm for circulating tumor cells (CTCs), enabling the development of size-based separation techniques, such as microfiltration, for specific diagnostic and therapeutic applications. Although cellular density is relatively constant among different circulating bioparticles, it allows for reliable density gradient centrifugation to isolate cells without altering their native state. Additionally, variations in EV surface charges (-6.3 to -45 mV) offer opportunities for electrophoretic and electrostatic separation methods. The distinctive mechanical properties of abnormal cells, compared to their normal counterparts, present an exceptional opportunity for diverse medical and biotechnological approaches. This review also aims to provide a holistic view of the current understanding of popular techniques in this domain that transcend conventional boundaries, focusing on early harvesting of malignant cells from body fluids, designing effective therapeutic options, cell targeting, and resonating with tissue and genetic engineering principles.

Advancing Diabetes Research: A Novel Islet Isolation Method from Living Donors.

Pancreatic islet isolation is critical for type 2 diabetes research. Although -omics approaches have shed light on islet molecular profiles, inconsistencies persist; on the other hand, functional studies are essential, but they require reliable and standardized isolation methods. Here, we propose a simplified protocol applied to very small-sized samples collected from partially pancreatectomized living donors. Islet isolation was performed by digesting tissue specimens collected during surgery within a collagenase P solution, followed by a Lympholyte density gradient separation; finally, functional assays and staining with dithizone were carried out. Isolated pancreatic islets exhibited functional responses to glucose and arginine stimulation mirroring donors' metabolic profiles, with insulin secretion significantly decreasing in diabetic islets compared to non-diabetic islets; conversely, proinsulin secretion showed an increasing trend from non-diabetic to diabetic islets. This novel islet isolation method from living patients undergoing partial pancreatectomy offers a valuable opportunity for targeted study of islet physiology, with the primary advantage of being time-effective and successfully preserving islet viability and functionality. It enables the generation of islet preparations that closely reflect donors' clinical profiles, simplifying the isolation process and eliminating the need for a Ricordi chamber. Thus, this method holds promises for advancing our understanding of diabetes and for new personalized pharmacological approaches.

[Study on Circulating Tumor Cell Detection System Based on Microfluidic Chip].

Objective: To achieve high throughput and high detection rate of circulating tumor cells (CTCs) in human peripheral blood, and to provide efficient and accurate early screening for cancer patients. Methods: A microfluidic chip with the integration of sorting, enrichment and detection was designed, and CTCs at the single cell level were detected by fluorescence detection system to obtain the number of CTCs in samples. Results: The peripheral blood samples after lysed red blood cells were used for 6 experiments. When the injection rate reached 0.2 mL/h, CTCs could reach the best detection rate of 78.6%, and the correlation coefficient within the group was above 0.8. Conclusion: CTCs detection system can achieve high detection rate and has good reliability, which can provide a reliable reference for clinical research in related fields.

High-throughput viable circulating tumor cell isolation using tapered-slit membrane filter-based chipsets in the differential diagnosis of ovarian tumors.

OBJECTIVE: To evaluate the diagnostic performance of circulating tumor cells (CTCs) using tapered-slit membrane filter (TSF)-based chipsets for the differential diagnosis of adnexal tumors. METHODS: A total of 230 women with indeterminate adnexal tumors were prospectively enrolled. The sensitivity, specificity, and accuracy of the CTC-detecting chipsets were analyzed according to postoperative pathological results and compared with those of cancer antigen (CA)-125 and imaging tests. RESULTS: Eighty-one (40.3%) benign tumors, 31 (15.4%) borderline tumors, and 89 (44.3%) ovarian cancers were pathologically confirmed. The sensitivity, specificity, and accuracy of CTC-detecting chipsets (75.3%, 58.0%, and 67.1%) for differentiating ovarian cancer from benign tumors were similar to CA-125 (78.7%, 53.1%, and 66.5%), but lower than CT/MRI (94.2%, 77.9%, and 86.5%). "CTC or CA125" showed increased sensitivity (91.0%) and "CTC and CA-125" revealed increased specificity (77.8%), comparable to CT/MRI. CTC detection rates in stage I/II and stage III/IV ovarian cancers were 69.6% and 81.4%, respectively. The sensitivity to detect high-grade serous (HGS) cancer from benign tumors (84.6%) was higher than that to detect non-HGS cancers (68.0%). CONCLUSION: Although the diagnostic performance of the TSF platform to differentiate between ovarian cancer and benign tumors did not yield significant results, the combination of CTC and CA-125 showed promising potential in the diagnostic accuracy of ovarian cancer.

Protocol for separating cancer cell subpopulations by metabolic activity using flow cytometry.

Cells, even from the same line, can maintain heterogeneity in metabolic activity. Here, we present a protocol, adapted for fluorescence-activated cell sorting (FACS), that separates resuspended cells according to their metabolic rate. We describe steps for driving lactate efflux, which produces an alkaline transient proportional to fermentative rate. This pH signature, measured using pH-sensitive dyes, identifies cells with the highest metabolic rate. We then describe a fluorimetric assay of oxygen consumption and acid production to confirm the metabolic contrast between subpopulations. For complete details on the use and execution of this protocol, please refer to Blaszczak et al.1.

Enhanced microfluidic multi-target separation by positive and negative magnetophoresis.

We introduce magnetophoresis-based microfluidics for sorting biological targets using positive Magnetophoresis (pM) for magnetically labeled particles and negative Magnetophoresis (nM) for label-free particles. A single, externally magnetized ferromagnetic wire induces repulsive forces and is positioned across the focused sample flow near the main channel's closed end. We analyze magnetic attributes and separation performance under two transverse dual-mode magnetic configurations, examining magnetic fields, hydrodynamics, and forces on microparticles of varying sizes and properties. In pM, the dual-magnet arrangement (DMA) for sorting three distinct particles shows higher magnetic gradient generation and throughput than the single-magnet arrangement (SMA). In nM, the numerical results for SMA sorting of red blood cells (RBCs), white blood cells (WBCs), and prostate cancer cells (PC3-9) demonstrate superior magnetic properties and throughput compared to DMA. Magnetized wire linear movement is a key design parameter, allowing device customization. An automated device for handling more targets can be created by manipulating magnetophoretic repulsion forces. The transverse wire and magnet arrangement accommodate increased channel depth without sacrificing efficiency, yielding higher throughput than other devices. Experimental validation using soft lithography and 3D printing confirms successful sorting and separation, aligning well with numerical results. This demonstrates the successful sorting and separating of injected particles within a hydrodynamically focused sample in all systems. Both numerical and experimental findings indicate a separation accuracy of 100% across various Reynolds numbers. The primary channel dimensions measure 100 µm in height and 200 µm in width. N52 permanent magnets were employed in both numerical simulations and experiments. For numerical simulations, a remanent flux density of 1.48 T was utilized. In the experimental setup, magnets measuring 0.5 × 0.5 × 0.125 inches and 0.5 × 0.5 × 1 inch were employed. The experimental data confirm the device's capability to achieve 100% separation accuracy at a Reynolds number of 3. However, this study did not explore the potential impact of increased flow rates on separation accuracy.

Separation of Activated T Cells Using Multidimensional Double Spiral (MDDS) Inertial Microfluidics for High-Efficiency CAR T Cell Manufacturing.

This study introduces a T cell enrichment process, capitalizing on the size differences between activated and unactivated T cells to facilitate the isolation of activated, transducible T cells. By employing multidimensional double spiral (MDDS) inertial sorting, our approach aims to remove unactivated or not fully activated T cells post-activation, consequently enhancing the efficiency of chimeric antigen receptor (CAR) T cell manufacturing. Our findings reveal that incorporating a simple, label-free, and continuous MDDS sorting step yields a purer T cell population, exhibiting significantly enhanced viability and CAR-transducibility (with up to 85% removal of unactivated T cells and approximately 80% recovery of activated T cells); we found approximately 2-fold increase in CAR transduction efficiency for a specific sample, escalating from ∼10% to ∼20%, but this efficiency highly depends on the original T cell sample as MDDS sorting would be more effective for samples possessing a higher proportion of unactivated T cells. This new cell separation process could augment the efficiency, yield, and cost-effectiveness of CAR T cell manufacturing, potentially broadening the accessibility of this transformative therapy and contributing to improved patient outcomes.

Protocol to develop human alveolar macrophage-like cells from mononuclear cells or purified monocytes for use in respiratory biology research.

Human alveolar macrophages are a unique myeloid subset critical for understanding pulmonary diseases and are difficult to access. Here, we present a protocol to generate human alveolar macrophage-like (AML) cells from fresh peripheral blood mononuclear cells or purified monocytes. We describe steps for cell isolation, incubation in a defined cocktail of pulmonary surfactant and lung-associated cytokines, phenotype analysis, and validation with human alveolar macrophages. We then detail procedures for quality control and technical readouts for monitoring microbial response. For complete details on the use and execution of this protocol, please refer to Pahari et al.1 and Neehus et al.2.

Repurposing discarded leukodepletion filters as a source of mononuclear cells for advanced in vitro research.

In light of advancements in the field of immuno-oncology, the demand for obtaining mononuclear cells for in vitro assays has surged. However, obtaining these cells from healthy donors remains a challenging task due to difficulties in donor recruitment and the requirement for substantial blood volumes. Here, we present a protocol for isolating peripheral blood mononuclear cells (PBMCs) from leukodepletion filters used in whole blood and erythrocytes by apheresis donations at the Hemonucleus of the Barretos Cancer Hospital, Brazil. The method involves rinsing the leukodepletion filters and subsequent centrifugation using a Ficoll-Paque concentration gradient. The isolated PBMCs were analyzed by flow cytometry, which allowed the identification of various subpopulations, including CD4+ T lymphocytes (CD45+CD4+), CD8+ T lymphocytes (CD45+CD8+), B lymphocytes (CD45+CD20+CD19+), non-classical monocytes (CD45+CD64+CD14-), classical monocytes (CD45+CD64+CD14+), and granulocytes (CD45+CD15+CD14-). In our comparative analysis of filters, we observed a higher yield of PBMCs from whole blood filters than those obtained from erythrocytes through apheresis. Additionally, fresh samples exhibited superior viability when compared to cryopreserved ones. Given this, leukodepletion filters provide a practical and cost-effective means to isolate large quantities of pure PBMCs, making it a feasible source for obtaining mononuclear cells for in vitro experiments. SUMMARY: Here, we provide a detailed protocol for the isolation of mononuclear cells from leukodepletion filters, which are routinely discarded at the Barretos Cancer Hospital's Hemonucleus.

Microchip for Immunomagnetic Sorting of Circulating Tumor Cells (CTCs).

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.

Greatly isolated heterogeneous circulating tumor cells using hybrid engineered cell membrane-camouflaged magnetic nanoparticles.

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.

Rapid Preparation of Living Drosophila Pupal Macrophages for Ex Vivo Imaging.

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.

Micropump integrated white blood cell separation platform for detection of chronic granulomatous disease.

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.

Unveiling heterogeneity in MSCs: exploring marker-based strategies for defining MSC subpopulations.

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.