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Magnetic-activated cell sorting

Magnetic-activated cell sorting (MACS) is a biotechnology technique used to isolate specific cell populations from heterogeneous mixtures by exploiting differences in cell surface markers, employing superparamagnetic microbeads conjugated to antibodies and high-gradient magnetic fields for separation.^[1] Developed in 1990 by Stefan Miltenyi and colleagues following the 1989 invention and founding of Miltenyi Biotec, MACS enables the gentle elution of target cells with minimal labeling impact, preserving their viability and functionality, distinguishing it from more stressful methods like fluorescence-activated cell sorting (FACS).^[1]^[2]^[3] The core principle of MACS relies on the specific binding of monoclonal antibodies coupled to nanoscale superparamagnetic particles (typically 50 nm in diameter) to target antigens on cell surfaces, followed by magnetophoresis in a high-gradient magnetic field.^[4]^[2] MACS supports both positive and negative selection strategies and can be performed manually or automated, offering high purity and recovery for various biomedical applications.^[2]

Introduction

Definition and Basic Concept

Magnetic-activated cell sorting (MACS) is a technique for isolating specific cell populations from heterogeneous mixtures based on their surface antigens, utilizing superparamagnetic nanoparticles conjugated to monoclonal antibodies that bind to target cell markers such as CD molecules.^[5]^[4] The core principle involves affinity-based labeling of target cells with these magnetic particles, followed by their retention in a high-gradient magnetic field while unlabeled cells pass through, enabling efficient separation without significant cell damage.^[5]^[6] This process relies on magnetophoresis, the directed migration of magnetically susceptible particles in a field gradient.^[5] Developed by Miltenyi Biotec in the early 1990s, MACS emerged as a gentle, high-throughput alternative to fluorescence-activated cell sorting (FACS), offering scalability for processing large cell numbers with preserved viability and functionality.^[5]^[4] Essential components include nano-sized superparamagnetic MicroBeads (approximately 50 nm in diameter), antigen-specific antibodies, and MACS separators equipped with columns that generate the required magnetic gradients.^[4]^[6]

Historical Development

Magnetic-activated cell sorting (MACS) was invented in 1990 by Stefan Miltenyi and colleagues, who introduced a novel system utilizing high-gradient magnetic separation (HGMS) for the efficient sorting of cells based on specific surface markers, as detailed in their seminal publication in Cytometry.^[7] This approach addressed limitations of fluorescence-activated cell sorting by enabling the rapid, high-throughput separation of large cell populations without the need for sophisticated flow cytometry equipment, marking a pivotal advancement in immunological research tools.^[7] Following the invention, Miltenyi Biotec, founded in 1989 to commercialize the technology, began marketing MACS systems in the early 1990s, which facilitated widespread adoption in research laboratories worldwide due to their simplicity, scalability, and compatibility with standard lab workflows.^[3] By the mid-1990s, the technology had been integrated into diverse applications, including stem cell isolation and immune cell phenotyping, underscoring its versatility and impact on biomedical research.^[3] In 2014, the FDA approved the CliniMACS CD34 Reagent System as a humanitarian use device for the ex vivo selection of CD34+ hematopoietic progenitor cells from donor apheresis products, aimed at reducing graft-versus-host disease in acute myeloid leukemia patients undergoing allogeneic stem cell transplantation.^[8] This endorsement solidified MACS's role in clinical cell therapy, promoting safer transplant outcomes through precise T-cell depletion.^[8] By the 2000s, MACS technology evolved from basic column-based setups to more integrated, automated platforms, enhancing reproducibility and throughput in immune cell analysis, as evidenced by over two decades of refinements that leveraged nanosized superparamagnetic particles for superior labeling efficiency. A 2010 review by Grützkau and Radbruch emphasized these advancements, noting how MACS had become indispensable for dissecting complex immune responses in health and disease.^[9] In November 2024, Miltenyi Biotec announced an agreement to supply the CliniMACS platform to Autolus Therapeutics for manufacturing AUCATZYL (obecabtagene autoleucel), an FDA-approved CAR T-cell therapy for relapsed/refractory B-cell ALL, further advancing MACS integration in approved cell therapies as of 2025.^[10]

Scientific Principles

Magnetic Labeling

Magnetic labeling in magnetic-activated cell sorting (MACS) involves the attachment of superparamagnetic iron oxide nanoparticles to specific target cells, enabling their subsequent isolation in a magnetic field. These nanoparticles, typically 50 nm in diameter and known as MACS MicroBeads, are composed of superparamagnetic iron oxide cores coated with biocompatible materials such as dextran to ensure minimal toxicity and cell perturbation.^[11]^[12] The beads are conjugated to monoclonal antibodies that recognize cell surface antigens, such as cluster of differentiation (CD) molecules (e.g., CD4 or CD8 on T cells), allowing for precise targeting of cell populations based on surface markers.^[4]^[13] Labeling can be achieved through direct or indirect methods to accommodate different experimental needs. In direct labeling, monoclonal antibodies specific to the target antigen are pre-conjugated to the MicroBeads, enabling a single-step binding process that simplifies the procedure and reduces non-specific interactions.^[14]^[12] Indirect labeling, by contrast, uses a two-step approach for signal amplification: first, unlabeled primary antibodies bind to the cell surface antigens, followed by the addition of MicroBeads conjugated to secondary antibodies (e.g., anti-mouse IgG or anti-biotin) that recognize the primary antibodies.^[4]^[12] This method is particularly useful for antigens expressed at low densities, as it enhances labeling efficiency through multivalent binding.^[12] The binding mechanism relies on high-affinity antigen-antibody interactions, where the monoclonal antibodies specifically recognize and attach to epitopes on the cell surface without requiring internalization, preserving cell integrity.^[11]^[13] Nanoparticle size and surface coatings are optimized for biocompatibility, with the nanoscale dimensions (around 50 nm) minimizing mechanical stress and phagocytic uptake by non-target cells, thus supporting high cell viability post-labeling.^[12]^[11] For applications requiring label-free cells after separation, releasable labeling options like REAlease technology from Miltenyi Biotec allow detachment of the beads via enzymatic cleavage using a recombinant enzyme that targets the antibody-bead linkage, yielding pure cells without residual magnetic particles.^[15] This approach maintains the specificity of antigen-antibody binding while enabling downstream analyses unhindered by persistent labels.^[15] Labeled cells are then amenable to high-gradient magnetic separation for isolation.^[14]

Separation Mechanism

Magnetic-activated cell sorting (MACS) relies on the principle of magnetophoresis, where magnetically labeled cells are subjected to a force in a non-uniform magnetic field, causing them to migrate toward regions of higher field strength.^[12] This motion, known as magnetophoresis, arises because the superparamagnetic particles attached to target cells become magnetized in the applied field, experiencing a net attractive force due to the field gradient.^[16] The separation is facilitated by high-gradient magnetic separation (HGMS), which utilizes columns packed with ferromagnetic spheres to generate intense magnetic field gradients, typically up to $10^4 T/m.^[12] These spheres concentrate the magnetic flux lines, creating localized high-gradient regions that amplify the force on labeled cells as the sample flows through the column under an external magnetic field.^[17] The magnetic force \mathbf{F}_m acting on a labeled cell is given by

\mathbf{F}_m = \frac{\chi V}{2 \mu_0} \nabla B^2,

where \mu_0 is the permeability of free space, \chi is the magnetic susceptibility of the particles, V is the volume of the magnetic material, and B is the magnetic flux density.^[18]^[12] This force originates from the interaction of the induced magnetic dipole moment in the superparamagnetic particles with the field gradient; the dipole aligns with the local field, and the spatial variation in field strength pulls the particles (and attached cells) toward stronger field regions, counteracting fluid drag and enabling selective retention.^[12] In positive selection, magnetically labeled target cells are retained by the column due to this force, while unlabeled cells pass through; in negative selection, the labeled unwanted cells are captured, allowing unlabeled target cells to elute freely.^[16] Superparamagnetic particles are essential, as they exhibit strong magnetization only in the presence of an external field and no remanence afterward, preventing agglomeration and ensuring reversible separation without residual attraction between particles.^[12]

Standard Procedure

Sample Preparation and Labeling

Sample preparation for magnetic-activated cell sorting (MACS) begins with isolating cells from primary tissues, blood, or cultured sources to create a single-cell suspension. Cells from adherent cultures are typically detached using enzymatic treatment, such as 0.25% trypsin-EDTA for 2-3 minutes at 37°C, followed by neutralization with culture medium and filtration through a 40 µm strainer to remove debris and aggregates.^[19] For blood or tissue samples, cells are obtained via density gradient centrifugation or mechanical dissociation, then pelleted by centrifugation at 300-600 × g for 10 minutes at 4°C.^[19] The resulting cell pellet is resuspended in a labeling buffer, commonly phosphate-buffered saline (PBS, pH 7.2) supplemented with 0.5% bovine serum albumin (BSA) and 2 mM EDTA, at a concentration of up to 10^8 cells per mL to minimize non-specific interactions and maintain cell viability.^[19]^[20] Magnetic labeling involves adding monoclonal antibody-coupled superparamagnetic microbeads specific to antigens on either target cells (for positive selection) or unwanted cells (for negative selection), either directly (pre-conjugated beads) or indirectly (via a secondary antibody). A typical dosage is 20 µL of microbeads (e.g., 50 nm MACS MicroBeads) per 10^7 cells, added to the cell suspension along with additional buffer (e.g., 80 µL per 10^7 cells) to achieve a final volume suitable for mixing.^[19]^[20] The mixture is gently agitated and incubated at 4°C for 15-30 minutes to allow specific binding, often in the dark to protect fluorochrome-conjugated components if present.^[19]^[20] This low-temperature incubation reduces metabolic activity and endocytosis, enhancing labeling specificity while preserving cell integrity.^[4]^[21] Following incubation, unbound microbeads and debris are removed through washing steps to ensure high specificity and prevent column clogging during separation. The labeled suspension is diluted with 1-2 mL of cold labeling buffer per 10^7 cells, centrifuged at 300 × g for 10 minutes at 4°C, and the supernatant is carefully aspirated.^[19]^[20] This process is repeated 1-2 times, with the final resuspension in 500 µL to 2 mL of buffer per 10^7 cells, depending on the downstream column capacity.^[19] Overloading with excess beads (beyond 20-50 µL per 10^7 cells) can lead to non-specific retention, so ratios are calibrated to target cell abundance.^[4] Prior to separation, quality checks assess cell viability and labeling efficiency to confirm suitability. Viability is evaluated using trypan blue exclusion, where >90% live cells (unstained) indicate a healthy suspension, or via flow cytometry with viability dyes like propidium iodide.^[22] Cell counts are performed using a hemocytometer or automated counter, and labeling specificity may be verified by flow cytometry detecting the target antigen, ensuring minimal off-target binding.^[22] These assessments help optimize yields, typically achieving 70-95% recovery of viable labeled cells.^[23]

Column-Based Separation

In column-based separation, the magnetically labeled cell suspension—prepared by incubating cells with antibody-coupled superparamagnetic MicroBeads—is loaded onto a MACS column positioned within a dedicated MACS separator, such as the MiniMACS or OctoMACS, which generates the required high-gradient magnetic field.^[14]^[6] For positive selection, unlabeled cells pass freely into the flow-through (negative fraction) and are collected, while labeled target cells are retained by high-gradient magnetic separation (HGMS) due to the amplified magnetic field within the column. In negative selection, labeled unwanted cells are retained, allowing unlabeled target cells to pass through as the positive fraction.^[13]^[4]^[17] To enhance separation efficiency and remove any residual non-target cells, the column is flushed multiple times with degassed buffer, typically at low flow rates of 1–5 mL/min to allow sufficient interaction time between the cells and the magnetic field without compromising cell viability.^[24]^[4] This step ensures high specificity by minimizing non-specific binding to the column matrix, which consists of spheres spaced approximately 20 times the diameter of a typical lymphocyte for gentle processing.^[6] MACS columns are available in various types optimized for different priorities; for instance, LS columns provide high purity suitable for downstream applications requiring enriched populations, with a capacity for up to 10^8 labeled cells (or 2 × 10^9 total cells) per separation, while MS columns emphasize high recovery for isolating rare cell types, accommodating up to 10^7 labeled cells (or 2 × 10^8 total cells).^[17]^[25] Standard setups using these columns in separators like the OctoMACS enable parallel processing of multiple samples, supporting throughputs of up to 10^8 cells per separation in routine laboratory protocols.^[14]^[13]

Elution and Collection

After the magnetic separation step, the column is removed from the magnet to allow elution. In positive selection, where magnetically labeled target cells are retained in the column due to the high-gradient magnetic field, the retained fraction is eluted. In negative selection, the target cells (unlabeled) are already collected in the flow-through, and the retained labeled unwanted cells may be eluted if needed for depletion confirmation. This release occurs primarily through gravity or by flushing the column with an elution buffer, such as phosphate-buffered saline (PBS) containing bovine serum albumin (BSA) and EDTA, using a plunger or syringe to gently dislodge the cells from the matrix without damaging them.^[14]^[19]^[4] The positive fraction from positive selection, consisting of the eluted labeled cells, is collected in a sterile tube, often in multiple sequential flushes to maximize recovery; for instance, initial gravity drainage followed by 1-2 buffer flushes of 500 µL per 10^7 cells can enhance yield while minimizing cell stress. To further improve purity, especially for applications requiring high enrichment like hematopoietic stem cell isolation, the eluted positive fraction may be reapplied to a second column for a second round of separation, achieving purities exceeding 95% for CD34+ cells in clinical settings.^[26]^[27] The negative fraction from positive selection, comprising unlabeled non-target cells, or the retained fraction from negative selection, is directly collected from the flow-through or post-elution, respectively, during the initial passage through the column under the magnetic field.^[4] Post-elution, collected fractions undergo washing via centrifugation at 300 × g for 10 minutes to remove residual buffer and beads, followed by resuspension in fresh medium, such as PBS or culture medium supplemented with fetal bovine serum. Yield and purity are then assessed, typically by flow cytometry; for example, CD34+ cell selections often yield >95% purity and >70% recovery from the starting population, confirming the effectiveness of the elution process.^[26]^[19] For storage, positively selected cells are resuspended in appropriate media for immediate downstream use, such as culturing at 37°C with 5% CO2, or cryopreserved in freezing medium containing dimethyl sulfoxide (DMSO) at -80°C or in liquid nitrogen to preserve viability for later applications. The negative fraction can similarly be processed and stored if needed for depletion-based studies.^[19]^[4]

Variants and Modifications

Selection Strategies

In magnetic-activated cell sorting (MACS), positive selection targets and retains cells expressing specific surface antigens by labeling them with superparamagnetic microbeads conjugated to antibodies, allowing their enrichment as they are captured in a high-gradient magnetic field within separation columns. This strategy is particularly effective for isolating rare cell populations, such as CD4+ T cells, where labeling with anti-CD4 antibodies achieves purities exceeding 95% while processing up to 10^9 cells in under 30 minutes. The original MACS system, developed using biotinylated antibodies and superparamagnetic particles, demonstrated over 100-fold enrichment of labeled cells without compromising viability or proliferation.^[1]^[2] Negative selection, in contrast, involves labeling and depleting undesired cell subsets, permitting the unlabeled target cells to pass through the magnetic column in the flow-through fraction, thereby enriching the remaining population indirectly. For instance, removing CD25+ regulatory T cells via anti-CD25 microbeads depletes these immunosuppressive cells, enriching effector T cell populations with yields up to 80% and minimal contamination from the targeted subset. This approach is advantageous when the target cells lack unique markers or when preserving their native state is critical, as demonstrated in protocols achieving greater than 90% purity for hematopoietic progenitors by depleting lineage-committed cells.^[2]^[28] Combined depletion-enrichment strategies employ sequential negative and positive selection steps to attain exceptionally high purity, often exceeding 99%, by first removing bulk unwanted cells and then positively isolating the targets. This multi-step process, such as initial depletion of CD19+ B cells followed by positive selection of CD4+CD25+ regulatory T cells, results in over 90% recovery of functional cells suitable for downstream applications like adoptive immunotherapy. Genetically modified CD4+ T cells have been enriched to 99% purity using such integrated labeling and separation, minimizing non-specific binding.^[2]^[29] A key biological consideration in these strategies is minimizing unintended activation of immune cells, as antibody-mediated labeling in positive selection can trigger intracellular signaling pathways, potentially altering cell function or phenotype, particularly in sensitive populations like T lymphocytes. Negative and combined approaches reduce this risk by avoiding direct labeling of targets, preserving immunosuppressive capacity in regulatory T cells isolated for clinical use.^[2]^[30] The choice of strategy depends on factors such as antigen density on target cells, which influences labeling efficiency in positive selection, and downstream assay requirements, where negative selection is favored for functional studies requiring untouched cells, while combined methods suit scenarios demanding both high purity and yield from heterogeneous samples.^[2]

Advanced and Emerging Techniques

Recent advancements in magnetic-activated cell sorting (MACS) have focused on reducing labeling artifacts and enhancing separation efficiency through innovative modifications. One such development is the REAlease technology, which employs recombinantly engineered antibody fragments with low binding affinity for reversible magnetic labeling.^[15] This approach allows for the removal of magnetic beads using a dedicated release reagent, yielding label-free cells without the need for proteolytic enzymes that could compromise cell viability or function.^[15] By avoiding permanent labeling, REAlease facilitates downstream applications such as functional assays or re-labeling with different markers, addressing a key limitation of traditional MACS protocols.^[31] Label-free MACS techniques have emerged to eliminate the need for antibody-based labeling altogether, leveraging intrinsic magnetic properties of cells or external media. Diamagnetophoresis, a form of negative magnetophoresis, exploits the repulsion of diamagnetic particles—such as unlabeled cells—in a paramagnetic ferrofluid medium under a magnetic field gradient.^[32] This method enables continuous, high-throughput separation based on differences in magnetic susceptibility, with applications in sorting mammalian cells like erythrocytes and leukocytes without surface modification. Ferrofluid-based systems have demonstrated purities exceeding 90% for specific cell types, offering a biocompatible alternative for sensitive samples.^[12] Microfluidic integrations represent another frontier, miniaturizing MACS for precise, continuous-flow processing. The LP CTC-iChip is an ultrahigh-throughput device that combines inertial focusing with magnetic deflection to deplete non-target cells from large volumes, achieving processing rates of over 6 billion nucleated cells per hour from leukapheresis products.^[33] This platform supports ex vivo manufacturing of therapies like CAR T cells by rapidly isolating rare populations with minimal shear stress.^[33] Such devices enhance scalability while maintaining high purity, typically recovering target cells at efficiencies above 80%.^[33] Innovations in targeting ligands have also advanced MACS specificity. Aptamer- and peptide-modified nanoparticles provide alternatives to antibodies, offering tunable affinity and reduced immunogenicity for magnetic labeling.^[12] A notable example is the use of coiled-coil peptides, as demonstrated by Shen et al., where cells are genetically engineered to express one peptide partner on their surface, enabling specific binding to complementary peptide-conjugated magnetic beads.^[34] This antigen-independent strategy achieves sorting efficiencies of up to 95% for diverse cell lines, with straightforward bead detachment post-separation, bypassing traditional enzymatic release.^[34] In vivo adaptations extend MACS beyond ex vivo settings, particularly for capturing rare circulating tumor cells (CTCs). The GILUPI CellCollector is an intravascular device inserted via peripheral vein access, featuring an EpCAM-functionalized wire that enriches CTCs directly from blood flow over 30 minutes.^[35] Clinical studies have validated its feasibility across lung cancer types, detecting CTCs in approximately 73% of patients with advanced disease and enabling molecular characterization without blood draw limitations.^[36] This approach increases CTC yield by orders of magnitude compared to standard venipuncture methods, supporting real-time monitoring in precision oncology.^[36]

Applications

Research Applications

Magnetic-activated cell sorting (MACS) is widely employed in immunology research to isolate specific T-cell subsets, such as CD8+ cytotoxic T cells, enabling detailed functional studies on their roles in immune responses. For instance, researchers have used MACS to purify CD8+ T cells from tumor-draining lymph nodes, allowing assessment of their antigen-specific reactivity and clonality through flow cytometry and tetramer staining, which revealed enhanced reactivation in response to anti-PD-1 therapy.^[37] This approach facilitates investigations into T-cell exhaustion and therapeutic modulation without contaminating other lymphocyte populations.^[38] In stem cell biology, MACS serves as a key method for enriching hematopoietic progenitors, particularly CD34+ cells, to support in vitro expansion for transplantation models and developmental studies. Purification via anti-CD34 magnetic beads achieves over 98% purity, removing undifferentiated cells and promoting multilineage differentiation potential when cultured with growth factors like SCF and FLT3L.^[39] Expanded CD34+ cells maintain long-term hematopoietic stem cell phenotypes, demonstrating robust myeloid and lymphoid output in nonhuman primate models.^[40] MACS-based techniques have advanced bacterial pathogen detection in research settings, particularly for food safety and microbiology, by enabling rapid isolation of low-abundance microbes like Salmonella from complex samples. A 2021 method combining biotin-exposed immunomagnetic separation with real-time PCR detected viable Salmonella Typhimurium at limits as low as 10 CFU/mL in milk, even amid high background bacteria, reducing detection time to under 9 hours.^[41] This sensitivity supports epidemiological studies and validation of contamination thresholds in agricultural products.^[42] In cancer research, MACS facilitates the isolation of rare circulating tumor cells (CTCs) from blood, providing material for genomic profiling to uncover tumor heterogeneity and resistance mechanisms. Enrichment using AutoMACS separators targets EpCAM-positive CTCs from melanoma patients, yielding sufficient purity for downstream next-generation sequencing of single cells and pooled populations to analyze mutations and copy number variations.^[43] Such applications have informed studies on metastatic progression and personalized therapy responses. For infectious disease research, MACS exploits the paramagnetic properties of hemozoin in malaria-infected erythrocytes to separate them from uninfected cells, aiding in parasite lifecycle analysis and diagnostic development. In Plasmodium falciparum cultures, MACS enriches late-stage infected red blood cells to 85-90% purity, enabling high-resolution imaging and phase-based classification for improved detection algorithms.^[44] This technique has been instrumental in studying host-pathogen interactions without reliance on morphological staining alone.

Clinical and Therapeutic Applications

Magnetic-activated cell sorting (MACS) plays a pivotal role in stem cell transplantation by enabling the positive selection of CD34+ hematopoietic stem cells using systems like CliniMACS, which facilitates bone marrow purging through T-cell depletion to mitigate graft-versus-host disease (GvHD) in allogeneic transplants.^[45] This approach has been integrated into clinical protocols, achieving high purity (median 93%) and yield (median 62%) of CD34+ cells from peripheral blood progenitor cells, supporting multiple transplantation cycles while reducing tumor cell contamination.^[46] In circulating tumor cell (CTC) isolation, the CellSearch system, approved by the FDA in 2004, employs immunomagnetic capture targeting epithelial cell adhesion molecule (EpCAM) to enumerate CTCs from peripheral blood, aiding in metastasis monitoring for metastatic breast, prostate, and colorectal cancers.^[47] This FDA-cleared technology provides prognostic information, with CTC counts ≥5 per 7.5 mL blood correlating with poorer progression-free and overall survival in breast cancer patients.^[48] For immunotherapy applications, MACS is utilized to purify chimeric antigen receptor (CAR) T cells and tumor-infiltrating lymphocytes (TILs) during manufacturing, ensuring high-purity populations for adoptive cell transfer.^[49] CliniMACS-based protocols enable the isolation and activation of CD3+ T cells for CAR-T production, supporting clinical-grade expansion with transduction efficiencies up to 50% in anti-CD19 therapies for B-cell malignancies.^[50] Similarly, MACS facilitates TIL enrichment from tumor digests, enhancing the yield of tumor-reactive CD8+ T cells for melanoma immunotherapy trials.^[51] In reproductive medicine, MACS is applied to select non-apoptotic sperm by targeting early apoptosis markers, reducing DNA fragmentation in assisted reproduction techniques such as intracytoplasmic sperm injection (ICSI). This method improves clinical outcomes, including higher fertilization rates and live birth rates, as demonstrated in studies comparing magnetically selected sperm to standard swim-up techniques.^[52] MACS contributes to pathogen removal in blood products by depleting bacteria or parasites, enhancing transfusion safety through magnetic bead-based negative selection.^[53] For instance, MACS columns effectively concentrate and purify Plasmodium falciparum-infected red blood cells while removing leukocytes, reducing malaria transmission risk in endemic areas with recovery rates exceeding 90%.^[53] This technique also targets bacterial contaminants in platelet units, lowering sepsis incidence in transfusions by isolating and removing infected cells prior to administration.^[54]

Advantages and Limitations

Key Advantages

Magnetic-activated cell sorting (MACS) offers high specificity and selectivity through the use of antibody-conjugated magnetic beads that target specific cell surface markers, enabling the isolation of desired cell populations with purities often exceeding 95%.^[55] This targeted approach minimizes non-specific binding and allows for both positive and negative selection strategies, making it particularly effective for enriching rare cell types such as stem cells or tumor cells from heterogeneous samples.^[56] Compared to fluorescence-activated cell sorting (FACS), MACS is more cost-effective and compact, as it requires no expensive lasers, fluidics systems, or fluorescence detection equipment, reducing overall setup and operational costs while fitting into standard laboratory spaces.^[57] The simplicity of the magnetic separation process, involving only a magnet and columns, further lowers maintenance needs and enables easier integration into workflows.^[56] MACS is gentle on cells, subjecting them to minimal physical stress during separation, which preserves high viability rates typically above 90% and maintains cellular function without significant perturbation.^[58] This biocompatibility stems from the use of superparamagnetic beads that do not retain magnetism once removed from the field, avoiding prolonged exposure that could damage delicate structures.^[5] The technique demonstrates excellent scalability, handling sample sizes from as few as 10^6 cells up to 10^9 cells per run, with high-throughput capabilities enhanced by multi-column configurations that process large volumes efficiently.^[56] Such flexibility supports both small-scale research and large-scale clinical preparations.^[23] MACS provides versatility across diverse sample types, including blood, bone marrow, and dissociated tissues, without relying on fluorescence, which broadens its applicability in scenarios where optical detection is impractical or unnecessary.^[56]

Principal Limitations

One principal limitation of magnetic-activated cell sorting (MACS) is its lower resolution compared to fluorescence-activated cell sorting (FACS), as MACS typically enables separation based on a single parameter, such as the presence or absence of a specific surface marker, rather than multiple parameters simultaneously. This bulk separation approach results in reduced purity for distinguishing closely related cell populations, particularly when relying on antibody-based labeling that is difficult to reverse.^[2] Additionally, labeling specificity can be compromised by non-specific antibody binding, potentially affecting separation accuracy.^[2] MACS can lead to potential cell activation or loss due to antibody binding and mechanical stresses during processing. Antibody-magnetic bead conjugates may trigger unwanted intracellular signaling cascades, activating cells and altering their function for downstream applications.^[2] Furthermore, passage through the separation column introduces shear forces and risks of non-specific binding or incomplete elution, contributing to cell loss; studies report losses of 7–9% in optimized protocols, though higher rates occur with suboptimal conditions or sensitive cell types. The cost of reagents represents another constraint, particularly for large-scale applications. Antibody-coupled magnetic bead kits and dedicated columns are expensive, driven by the production of animal-derived antibodies and batch variability, limiting accessibility for routine or high-volume use.^[2] Overloading the separation column poses a risk of reduced efficiency and increased cell loss. Standard columns have a limited capacity, typically handling up to 2 × 10^8 total cells (with 10^7 labeled cells), necessitating multiple runs for larger samples and complicating scalability.^[59] In point-of-care devices, MACS faces sample volume constraints, as many platforms process only a few microliters, restricting applicability for rare cell detection or field-based diagnostics that require larger inputs.^[2]

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Ultrahigh-throughput magnetic sorting of large blood volumes for ...
Jul 8, 2020 · We describe an ultrahigh-throughput microfluidic chip, LP CTC-iChip, that rapidly sorts through an entire leukapheresis product of over 6 billion nucleated ...Missing: LPCTC- MACS
[33]
Magnetic-Activated Cell Sorting Using Coiled-Coil Peptides
Mar 3, 2021 · Magnetic-activated cell sorting (MACS) is an affinity-based technique used to separate cells according to the presence of specific markers.Introduction · Experimental Section · Supporting Information · References<|control11|><|separator|>
[34]
A Review of Circulating Tumour Cell Enrichment Technologies - PMC
Feb 26, 2021 · The GILUPI CellCollector is a novel in vivo CTC enrichment device designed to overcome the limitations faced by in vitro devices in trying ...<|control11|><|separator|>
[35]
In vivo isolation of circulating tumor cells in patients with different ...
Mar 4, 2021 · The present study examined the clinical validity and feasibility of a new medical device for the in vivo isolation of CTCs from the blood of patients with ...Missing: MACS | Show results with:MACS
[36]
Reactivation of low avidity tumor-specific CD8+ T cells associates ...
Aug 16, 2023 · Total CD8+ T cells were purified using CD8 magnetic-activated cell sorting ... CD8+ T cell subsets isolated from the regressing and ...
[37]
https://pmc.ncbi.nlm.nih.gov/articles/PMC10432680/
[38]
Efficient generation, purification, and expansion of CD34+ ... - NIH
Treatment with PGE2 and SR1 enhances hematopoietic progenitor emergence and expansion. We next tested whether PGE2 or SR1, which activate downstream ...
[39]
https://pmc.ncbi.nlm.nih.gov/articles/PMC3460690/
[40]
Biotin exposure-based immunomagnetic separation coupled with ...
Mar 11, 2021 · In this study, we established a rapid and sensitive method for the detection of viable Salmonella Typhimurium, Staphylococcus aureus, and ...
[41]
https://pubmed.ncbi.nlm.nih.gov/33715855/
[42]
Next-generation Sequencing (NGS) Analysis on Single Circulating ...
Melanoma cells were first enriched using an AutoMACS® (Miltenyi) cell separator and then isolated as single and pooled CTCs by DEPArray™ System (Silicon ...Missing: MACS | Show results with:MACS
[43]
Enhanced detection of malaria infected red blood cells through ...
Aug 21, 2025 · Infected RBCs are then isolated via magnetic-activated cell sorting (MACS), a technique developed by Miltenyi Biotec. The MACS-purified samples ...
[44]
Allogeneic Stem Cell Transplantation with CD34+ Cell Selection - NIH
T cell depletion through positive selection of CD34+ cells has emerged as a promising strategy to reduce acute and chronic GvHD in these patients.
[45]
CliniMACS CD34-selected cells to support multiple cycles ... - PubMed
Results: PBPC from 10 patients underwent CD34-selection with a resulting median purity of 93% (range: 76-98%) and yield of 62% (range: 16-93%). Of the 10 ...
[46]
[PDF] K052191 - REVIEW MEMORANDUM - FDA
The CellSearch™ Circulating Tumor Cell Kit is intended for the enumeration of circulating tumor cells (CTC) of epithelial origin (CD45-, EpCAM+, and.Missing: approval | Show results with:approval
[47]
Clinical manufacturing of CAR T cells: foundation of a promising ...
Jun 15, 2016 · In this review, we highlight the cGMP manufacturing platforms and the quality control requirements for clinical-grade CAR-T cells in early phase clinical ...T-Cell Source · T-Cell Activation · Clinical Car-T Cell...
[48]
CAR T cell activation - Miltenyi Biotec
To demonstrate the state-of-the-art panel, CAR T cells were generated within 12 days using the CliniMACS Prodigy® T Cell Transduction process. CAR T cell ...
[49]
Clinical-grade manufacturing of TILs for therapy - Miltenyi Biotec
Automate and standardize your tumor-infiltrating lymphocyte (TIL) manufacturing workflow for TIL therapy with the CliniMACS Prodigy® Tumor-Reactive T Cell ...Missing: purification | Show results with:purification
[50]
In vivo isolation of circulating tumor cells in patients with different ...
In vivo CTC isolation. The CellCollector consists of a 160-mm sterile steel wire with a 20-mm functionalized tip containing epithelial-cell adhesion molecule ( ...Missing: magnetic | Show results with:magnetic
[51]
One-step concentration of malarial parasite-infected red blood cells ...
The magnetic removal of WBCs using a commercial LD column (MACS) was evaluated as a new method for concentrating and purifying PRBCs. To compare this method ...
[52]
Magnetic-Activated Cell Sorting - an overview | ScienceDirect Topics
MACS is a magnetic-based cell separation using magnetically susceptible beads bound to antibodies to isolate specific cells in a magnetic field.
[53]
Efficient immunoselection of cytolytic effectors with a magnetic cell ...
The purity obtained for positive (CD3+) cell sorting with the MACS was higher than 95%. The purity of negative (CD3-) cell fraction was more variable, but ...
[54]
Magnetic-Activated Cell Sorting - an overview | ScienceDirect Topics
Magnetic activated cell sorting (MACS) is a technique that captures circulating tumor cells (CTCs) by immunolabeling them with superparamagnetic particles, ...
[55]
https://pubmed.ncbi.nlm.nih.gov/8516559/
[56]
Adult Neural Stem Cell Analysis Cocktail Kit, anti-mouse
A workflow protocol is established for fast and pure isolation of NSCs (total process time 2h, purity >95%, viability >93%). Detailed product information.
[57]
High-throughput, low-loss, low-cost, and label-free cell separation ...
Apr 13, 2017 · FACS can sort up to 50,000 cells per second, with higher rates achievable at the cost of purity. Finally, magnetically activated cell sorting ( ...
[58]
Development of the “Three-step MACS”: a Novel Strategy for ... - NIH
The Dead Cell Removal Kit (Miltenyi Biotec) was used to remove dead cells from cell samples, following the manufacturer's instructions. Briefly, MACS was ...