Cell Separation Research Articles

Nonmonotonous Translocation Dynamics of Highly Deformable Particles across Channels.

The translocation dynamics of cells and particles through geometric constrictions are critical in biological and biomedical processes from splenic filtration to tumor metastasis. While particle stiffness plays a key role, its role in highly nonequilibrium states remains poorly understood. Here, we present a multiscale model to investigate the impact of particle stiffness on the translocation dynamics in microfluidic channels. We find that semielastic particles exhibit superior translocation capabilities compared to both softer and more rigid particles, with a nonmonotonic stiffness dependence observed for highly deformable particles. Additionally, we identify crossover behaviors in translocation time driven by variations in the flow rate, particle size, and particle-plate interactions. Excessive particle deformation significantly regulates these dynamics, with stiffness-induced shape transitions from pancake-like to ellipsoidal forms, controlling frictional forces at the particle-channel interface and the sieve plate. The balance between these forces explains the observed nonmonotonic translocation dynamics. Our work provides insights into the relationship between particle deformability and flow dynamics, highlighting the importance of elasticity in translocation behavior. These findings have implications for designing microfluidic devices for efficient separation and analysis of cells with varying elasticities, advancing applications in human health diagnostics.

Novel insights into taxane pharmacology: An update on drug resistance mechanisms, immunomodulation and drug delivery strategies.

Novel insights into taxane pharmacology: An update on drug resistance mechanisms, immunomodulation and drug delivery strategies.

Anisotropic Porous Iron-Based Nanoparticles through Two-Step Hydrothermal and Hydrogen-Based Reduction: Enhanced Magnetic Performance for Potential Biomedical Applications.

Iron-based nanoparticles have emerged as promising candidates for diverse biomedical applications, including cell separation, targeted drug delivery, hyperthermia therapy, and magnetic resonance imaging. This study reports the scalable synthesis of high-magnetization iron-based nanoparticles with controlled anisotropic shapes, achieved via a two-step process. Hematite nanoparticles, featuring nanocube, nanoellipse, and nanoneedle morphologies, were synthesized through the hydrolysis of ferric chloride in the presence of ammonium dihydrogen phosphate, with the morphology precisely tuned by adjusting reagent concentrations. These hematite nanoparticles were subsequently reduced in a hydrogen-based direct reduction at 480 °C, yielding iron-magnetite nanocomposites that retained their anisotropic shapes, exhibited significant porosity, and achieved an exceptional saturation magnetization of 207 emu/g - approximately 150% higher than conventional magnetite nanoparticles. Comprehensive characterization via SQUID magnetometry, Mössbauer spectroscopy, Rietveld refinement of X-ray diffraction data, and XPS for surface analysis confirmed the formation of metallic iron nanoparticles covered by a magnetite shell. Biocompatibility studies demonstrated the biocompatibility of these nanoparticles across a wide concentration range, underscoring their suitability for biomedical applications.

Potential of MSCA1 for isolating osteogenic cells in a chondrocyte population.

The aim of the present study was to elucidate the utility of Mesenchymal Stem Cell Antigen-1 (MSCA1) (tissue nonspecific alkaline phosphatase (TNAP)) as a potential marker for purification of human chondrocyte fraction with less heterogenous phenotype from those with osteogenic properties. Chondrocytes were isolated from human osteoarthritis cartilage and sorted according to MSCA1 expression by MACS (Magnetic-activated cell sorting) and FACS (Fluorescence-activated cell sorting) techniques (MSCA1high and MSCA1low), analyzed for gene expression, osteogenic and adipogenic differentiation capacities, and were compared between the sorted populations. Gene expression analyses revealed upregulation in osteogenic genes (ALPL and RUNX2) and significantly lower expression of chondrocyte-specific genes (COL2A1, SOX9 and MIA) in sorted MSCA1high, as compared to MSCA1low. Expression of Aldehyde dehydrogenase isoform gene ALDH1A2 was enriched in MSCA1low chondrocytes. Stronger osteogenic differentiation was observed in MSCA1high, as compared to the unsorted cells, and particularly, MSCA1low chondrocytes. Expression of MSCA1 in human chondrocytes is biased in their commitment to the osteogenic lineage. We demonstrate that MSCA1 can be used as a marker for separation of cells, prone to differentiate towards osteogenic lineage from those, retaining chondrogenic phenotype. This may allow enrichment of a chondrogenic population and separation from undesirable osteoprogenitors.

4,4′-methylenebis(2-chloroaniline) induces chromosome aneuploidy associated with premature chromatid separation in mammalian cells: A possible carcinogenic mechanism

4,4′-methylenebis(2-chloroaniline) induces chromosome aneuploidy associated with premature chromatid separation in mammalian cells: A possible carcinogenic mechanism

Dependence of acoustophoretic aggregation on the impedance of microchannel's walls

Dependence of acoustophoretic aggregation on the impedance of microchannel's walls

Double difference accumulation SERS strategy for rapid separation and detection of probiotic Bacillus endospores and vegetative cells

Double difference accumulation SERS strategy for rapid separation and detection of probiotic Bacillus endospores and vegetative cells

Implications of packed red bloods cells production and transfer on post transfusion hemoglobin increase.

Implications of packed red bloods cells production and transfer on post transfusion hemoglobin increase.

Ultra-low extracorporeal volume microfluidic leukapheresis is safe and effective in a rat model

Leukapheresis is a potentially life-saving therapy for children with symptomatic hyperleukocytosis. However, the standard centrifugation-based approach exposes pediatric patients to significant complications due to its large extracorporeal volume, high flow rates, and considerable platelet loss. Here, we tested whether performing cell separation with a high-throughput microfluidic technology could alleviate these limitations. In vitro, our microfluidic devices removed ~85% of large leukocytes and ~90% of spiked leukemic blasts from undiluted human whole blood, while minimizing platelet losses. Multiplexed devices connected in parallel allowed for faster, clinically relevant flow rates in vitro with no difference in leukocyte collection efficiency. When connected to Sprague-Dawley rats, the devices removed large leukocytes with ~80% collection efficiency, reducing the leukocyte count in recirculating blood by nearly half after a 3-hour procedure. Evaluation of plasma biomarkers and end-organ histology revealed no adverse effects compared to sham control. Overall, our study suggests that microfluidics-based leukapheresis is safe and effective at selectively removing leukocytes from circulation, with separation performance sufficiently high to ultimately enable low extracorporeal volume leukapheresis in children.

Abstract B023: Enhancing the efficacy of oncolytic virus immunotherapy using eicosapentaenoic acid in preclinical cancer models

Abstract Introduction Despite significant advances in cancer immunotherapy, response rates remain limited and variable across patients and cancer types. Oncolytic viruses (OVs) are cancer immunotherapeutic agents, which kill cancer cells and promote an anti-tumor immune response. However, upregulation of immune evasion pathways, such as COX-2 mediated prostaglandin E2 (PGE2) signaling by cells in the tumor microenvironment (TME), including tumor associated macrophages (TAMs), can hinder their immunotherapeutic potential. This study investigated whether combining OVs with eicosapentaenoic acid (EPA), an omega-3 polyunsaturated fatty acid with anti-inflammatory properties, including inhibition of PGE2 production, could enhance OV anti-cancer activity. Methods Balb/c mice were injected with colorectal CT26 cells and treated with OV (reovirus), EPA, or a combination of both reovirus/EPA, and tumor growth was monitored. Upon sacrifice, immune cell infiltration within harvested tumors was assessed by immunohistochemistry (IHC). PGE2 and cytokine production was determined by ELISA while MTT assays were employed to measure cell growth inhibition, in vitro. Co-culture of cancer cells with human peripheral blood mononuclear cells (PBMCs) was used to generate tumor-associated macrophages (TAMs), which were then isolated by magnetic bead CD14+ cell separation Results Assessment of EPA and reovirus in vivo suggested that the combination treatment reduced tumor volume more than the single agent treatments and confirmed that EPA significantly reduced PGE2 production. The combination treatment strategy also significantly inhibited the production CXCL9 and reduced the percentage of arginase-1 positive cells, a marker of TAMs. In vitro, EPA supplementation did not alter abrogate reovirus direct oncolysis and furthermore potentially enhanced reovirus oncolysis of CT26 cells. Additionally, EPA supplementation hindered PGE2 production and significantly inhibited the in vitro generation of TAMs by 50-90%. Conclusion These results suggest that EPA could render the TME less immunosuppressive without compromising on the direct oncolytic effect of the virus, ultimately enhancing OV-based immunotherapy. Citation Format: Nametso Koobotse Khumo, Milene Volpato, Fiona Errington-Mais. Enhancing the efficacy of oncolytic virus immunotherapy using eicosapentaenoic acid in preclinical cancer models [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B023.

Abstract B004: NK cells demonstrate robust cytotoxicity against ovarian high-grade serous carcinoma in 2D and 3D co-culture models

Abstract Background: High-grade serous carcinoma (HGSC) is the most aggressive form of ovarian cancer. Relapse is frequent following current standard treatment combining surgery and platinum-based chemotherapy, leading chemoresistance that is ultimately fatal. Novel treatment options, including immunotherapy, are required for HGSC patients. Natural Killer (NK) cells have shown exceptional potential as an “off-the-shelf" adoptive cell therapy against various malignancies. However, there have been few studies specifically examining the interactions of NK cells from diverse sources against HGSC cells. In this study, we optimized a NK/HGSC co-culture system in both 2D monolayer and 3D spheroid models, tested the cytotoxicity of NK92 cell line on both established and primary HGSC cell lines, and subsequently explored the cytotoxicity of umbilical cord blood (UCB)-derived NK cells, harvested from five donors, against primary HGSC cultures in vitro. Method: NK92 cells, at effect-to-target (E:T) ratios of 1:1, 3:1 and 10:1, were added to established (PEO1, PEO4, OVCAR4) and primary (ASC-24-003, Br2-043, Br2-205) HGSC cells in 2D and 3D. Cytotoxicity was measured using both IncuCyte live-cell imaging and cell viability assays. To generate primary NK cells, UCB from 5 healthy donors was processed by Ficoll-Paque layering to isolate mononuclear cells (MNCs). Cord MNCs were depleted of CD3+ cells using magnetic cell separation. To expand UCB-NK, CD3- Cord MNCs were cultured in RPMI 1640 media, 10% FBS and 100IU IL-2 with sub-lethally irradiated feeder cell line K562 engineered to express membrane bound IL-21 for a minimum of 14 days. The expanded UCB-derived NK cells were added to HGSC cell lines (PE20-001, ASC22-030, Br2-043) at E:T ratios of 1:1, 3:1 and 10:1. The cytotoxic effects were measured by cell death assay. Result: NK92 cells at higher E:T ratios (3:1,10:1) significantly reduced viability of established cell lines growing in 2D. The viability of platinum-resistant PEO4 also had significantly decreased at E:T ratio of 1:1. The viability of all primary cells decreased significantly at all E:T ratios in 2D. Live cell imaging assays yielded comparable results using cell confluency. In the UCB-derived NK cell experiments, cells from every donor induced cell death in all HGSC cell lines at all E:T ratios in 2D. In 3D, every donor again induced cell death at all E:T ratios with the exception of PE20-001 cell line treated with donor 1 NK cells at 1:1 ratio. Cytotoxic effects of UCB-derived NK cells were largely induced within 24 hours. Donor 4 and 5 NK cells showed superior cytotoxicity to NK92 cells when added in the same E:T ratio at the same experiment setting. Conclusion: These results demonstrate the potential of adoptive NK cell therapy, especially with donor-derived NK cells, in treating HGSC cell models. Further in-vitro studies exploring inter-donor variability and spheroid penetration, in-vivo studies assessing the efficacy of donor NK in mouse models and genetic arming of stimulatory receptors are under way. Citation Format: Deyi Yang, Darren P Ennis, Preethika Mahalingam, Joanna Burr, Hugh J M Brady, Iain A McNeish. NK cells demonstrate robust cytotoxicity against ovarian high-grade serous carcinoma in 2D and 3D co-culture models [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B004.

Abstract B004: NK cells demonstrate robust cytotoxicity against ovarian high-grade serous carcinoma in 2D and 3D co-culture models

Abstract Background: High-grade serous carcinoma (HGSC) is the most aggressive form of ovarian cancer. Relapse is frequent following current standard treatment combining surgery and platinum-based chemotherapy, leading chemoresistance that is ultimately fatal. Novel treatment options, including immunotherapy, are required for HGSC patients. Natural Killer (NK) cells have shown exceptional potential as an “off-the-shelf" adoptive cell therapy against various malignancies. However, there have been few studies specifically examining the interactions of NK cells from diverse sources against HGSC cells. In this study, we optimized a NK/HGSC co-culture system in both 2D monolayer and 3D spheroid models, tested the cytotoxicity of NK92 cell line on both established and primary HGSC cell lines, and subsequently explored the cytotoxicity of umbilical cord blood (UCB)-derived NK cells, harvested from five donors, against primary HGSC cultures in vitro. Method: NK92 cells, at effect-to-target (E:T) ratios of 1:1, 3:1 and 10:1, were added to established (PEO1, PEO4, OVCAR4) and primary (ASC-24-003, Br2-043, Br2-205) HGSC cells in 2D and 3D. Cytotoxicity was measured using both IncuCyte live-cell imaging and cell viability assays. To generate primary NK cells, UCB from 5 healthy donors was processed by Ficoll-Paque layering to isolate mononuclear cells (MNCs). Cord MNCs were depleted of CD3+ cells using magnetic cell separation. To expand UCB-NK, CD3- Cord MNCs were cultured in RPMI 1640 media, 10% FBS and 100IU IL-2 with sub-lethally irradiated feeder cell line K562 engineered to express membrane bound IL-21 for a minimum of 14 days. The expanded UCB-derived NK cells were added to HGSC cell lines (PE20-001, ASC22-030, Br2-043) at E:T ratios of 1:1, 3:1 and 10:1. The cytotoxic effects were measured by cell death assay. Result: NK92 cells at higher E:T ratios (3:1,10:1) significantly reduced viability of established cell lines growing in 2D. The viability of platinum-resistant PEO4 also had significantly decreased at E:T ratio of 1:1. The viability of all primary cells decreased significantly at all E:T ratios in 2D. Live cell imaging assays yielded comparable results using cell confluency. In the UCB-derived NK cell experiments, cells from every donor induced cell death in all HGSC cell lines at all E:T ratios in 2D. In 3D, every donor again induced cell death at all E:T ratios with the exception of PE20-001 cell line treated with donor 1 NK cells at 1:1 ratio. Cytotoxic effects of UCB-derived NK cells were largely induced within 24 hours. Donor 4 and 5 NK cells showed superior cytotoxicity to NK92 cells when added in the same E:T ratio at the same experiment setting. Conclusion: These results demonstrate the potential of adoptive NK cell therapy, especially with donor-derived NK cells, in treating HGSC cell models. Further in-vitro studies exploring inter-donor variability and spheroid penetration, in-vivo studies assessing the efficacy of donor NK in mouse models and genetic arming of stimulatory receptors are under way. Citation Format: Deyi Yang, Darren P Ennis, Preethika Mahalingam, Joanna Burr, Hugh J M Brady, Iain A McNeish. NK cells demonstrate robust cytotoxicity against ovarian high-grade serous carcinoma in 2D and 3D co-culture models [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B004.

Multivalent afadin interaction promotes IDR-mediated condensate formation and junctional separation of epithelial cells.

Multivalent afadin interaction promotes IDR-mediated condensate formation and junctional separation of epithelial cells.

Force Map-Enhanced Segmentation of a Lightweight Model for the Early Detection of Cervical Cancer.

Background/Objectives: Accurate and efficient segmentation of cervical cells is crucial for the early detection of cervical cancer, enabling timely intervention and treatment. Existing segmentation models face challenges with complex cellular arrangements, such as overlapping cells and indistinct boundaries, and are often computationally intensive, which limits their deployment in resource-constrained settings. Methods: In this study, we introduce a lightweight and efficient segmentation model specifically designed for cervical cell analysis. The model employs a MobileNetV2 architecture for feature extraction, ensuring a minimal parameter count conducive to real-time processing. To enhance boundary delineation, we propose a novel force map approach that drives pixel adjustments inward toward the centers of cells, thus improving cell separation in densely packed areas. Additionally, we integrate extreme point supervision to refine segmentation outcomes using minimal boundary annotations, rather than full pixel-wise labels. Results: Our model was rigorously trained and evaluated on a comprehensive dataset of cervical cell images. It achieved a Dice Coefficient of 0.87 and a Boundary F1 Score of 0.84, performances that are comparable to those of advanced models but with considerably lower inference times. The optimized model operates at approximately 50 frames per second on standard low-power hardware. Conclusions: By effectively balancing segmentation accuracy with computational efficiency, our model addresses critical barriers to the widespread adoption of automated cervical cell segmentation tools. Its ability to perform in real time on low-cost devices makes it an ideal candidate for clinical applications and deployment in low-resource environments. This advancement holds significant potential for enhancing access to cervical cancer screening and diagnostics worldwide, thereby supporting broader healthcare initiatives.

The Latest Advances in Microfluidic DLD Cell Sorting Technology: The Optimization of Channel Design.

Cell sorting plays a crucial role in both medical and biological research. As a key passive sorting technique in the field of microfluidics, deterministic lateral displacement (DLD) has been widely applied to cell separation and sorting. This review aims to summarize the latest advances in the optimization of channel design for microfluidic DLD cell sorting. First, we provide an overview of the design elements of microfluidic DLD cell sorting channels, focusing on key factors that affect separation efficiency and accuracy, including channel geometry, fluid dynamics, and the interaction between cells and channel surfaces. Subsequently, we review recent innovations and progress in channel design for microfluidic DLD technology, exploring its applications in biomedical fields and its integration with machine learning. Additionally, we discuss the challenges currently faced in optimizing channel design for microfluidic DLD cell sorting. Finally, based on existing research, we make a summary and put forward prospective views on the further development of this field.

A novel peptidoglycan deacetylase modulates daughter cell separation in E. coli.

Bacteria surround their cell membrane by the essential peptidoglycan (cell wall) layer to prevent bursting open due to their turgor. During cell division, bacteria produce a septum at midcell, which must be cleaved for daughter cells to separate. Here, we report the identification of a new enzyme, SddA, that modifies a particular type of peptidoglycan material, denuded glycan chains released during the splitting of septal peptidoglycan for daughter cell separation, in the Gram-negative Escherichia coli . We propose a model in which SddA modulates a switch in the septal peptidoglycan splitting, ensuring splitting is activated from the cell membrane in the early stages of cell division and from the outer membrane in the late stages.

Enhanced Particle Trap: Design and Simulation of Pillar-Based Contactless Dielectrophoresis Microfluidic Devices.

Contactless and conventional dielectrophoresis (DEP) microfluidic devices are extensively utilized in lab-on-a-chip applications, particularly for cell isolation and analysis. Nonetheless, these devices typically operate at low throughput and require high applied voltages, posing limitations for microfluidic cell isolation and separation. Addressing these challenges, this study explores the utilization of diverse micro-pillar geometries within the microfluidic device to augment THP-1 cell trapping efficiency numerically using FEM modeling. Furthermore, the simulations examine the influence of pillar gap and quantity on cell trapping efficiency in a contactless DEP device. Notably, elliptical pillars demonstrate superior cell trapping efficiency at elevated flow rates compared to alternative configurations, making the microchip more amenable for high-throughput cell separation, trapping, and isolation applications. Remarkably, employing elliptical pillars in a contactless DEP microfluidic chip yields nearly 100% cell trapping efficiency at higher flow rates. Ellipse configuration showed 122% higher cell trap efficiency at the maximum flowrate compare to the previous study with circular configuration. Additionally, it is observed that reducing the gap between pillars correlates with enhanced cell trapping efficiency. Simulation outcomes indicate that employing two rows of elliptical pillars with a 40-µm gap achieves optimal performance. The findings of this investigation underscore the importance of pillars in contactless DEP devices and provide valuable insights for future designs of such microfluidic devices.

A Cascaded Chip for the High-Purity Capture and Distinguishing Detection of Phenotypic Circulating Tumor Cells in Colon Cancer.

The low abundance, complex phenotypes, and need for sophisticated blood preprocessing pose substantial obstacles to the clinical implementation of circulating tumor cells (CTCs). Herein, we constructed a cascaded PMMA chip-based platform for the separation of CTCs from other cells within blood samples, as well as distinguishing the detection of epithelial and mesenchymal CTCs. The primary physical separation chip (PS-Chip) focused and sorted CTCs from whole blood via Dean flow fractionation (DFF) according to size differences between CTCs and other blood cells, being capable of eliminating approximately 93.7% of red blood cells (RBCs) and 68.4% of white blood cells (WBCs) from whole blood while maintaining a CTC recovery rate of around 90%. Subsequently, to further purify the isolated CTCs in the upstream, a partitioned immunoaffinity capture and detection chip (PICD-Chip) featuring with two independent chambers (Zone 1, Zone 2) was designed, each of which was premodified with Gel-GO/E/V-Apt complexes that specifically recognize CTCs with distinct phenotypes, enabling further separation of residual blood cells from the upstream isolation. Upon the subsequent introduction of two detection probes, namely EpCAM and vimentin aptamer-modified mesoporous Pt nanoparticles (mPtNPs/E/V-Apt), into Zone 1 and Zone 2, respectively, heterogeneous CTCs ranging from 5 to 200/mL captured within two chambers were distinguished and quantified utilizing the exceptional peroxidase activity of mPtNPs. The integrated approach of efficient enrichment and differentiation detection of phenotypic CTCs under the requirement of high purity has enabled the successful application of the cascaded chip in the diagnosis of colon cancer patients at different stages.

Mapping T cell dynamics to molecular profiles through behavior-guided transcriptomics.

The rise of cellular immunotherapy for cancer treatment has led to the utilization of immune oncology cocultures to simulate T cell interactions with cancer cells for assessing their antitumor response. Previously, we developed BEHAV3D, a three-dimensional live imaging platform of patient-derived tumor organoid (PDO) and engineered T cell cocultures, that analyzes T cells' dynamics to gain crucial insights into their behavior during tumor targeting. However, live imaging alone cannot determine the molecular drivers behind these behaviors. Conversely, single-cell RNA sequencing (scRNA-seq) allows researchers to analyze the transcriptional profiles of individual cells but lacks spatio-temporal resolution. Here we present an extension to the BEHAV3D protocol, called Behavior-Guided Transcriptomics (BGT), for integration of T cell live imaging data with single-cell transcriptomics, enabling analysis of gene programs linked to dynamic T cell behaviors. BGT uses live imaging data processed by BEHAV3D to guide the experimental setup for cell separation based on their PDO engagement levels subsequently followed by fluorescence-activated cell sorting and scRNA-seq. It then integrates in silico simulations of these experiments to computationally infer T cell behavior on scRNA-seq data, uncovering new biomarkers for both highly functional and ineffective T cells, that could be exploited to enhance therapeutic efficacy. The protocol, designed for users with fundamental cell culture, imaging and programming skills, is readily adaptable to diverse coculture settings and takes one month to perform.

Magnetic Cell Separation Based on Protein Nanoparticles Mediating the Interaction between Magnetic Particles and Target Cells.

Isolation of specific cells from biological samples is an important aspect of various biological research and diagnostic applications. Magnetic separation using magnetic particles (MPs) allows for easy and specific isolation of the target cells. However, depending on the target cell antigen, biological ligands, such as antibodies, must be modified or altered on MPs. Additionally, further biological evaluation of isolated cells requires the removal of MPs from cells by the enzymatic degradation of the biological ligands. In this study, we designed a magnetic cell separation system in which temperature-responsive protein nanoparticles mediated the interaction between target cells and MPs, achieving the easy changeability of biological ligands, removal of MPs by cooling, and effective cell isolation. The protein nanoparticles were thermally responsively formed from fusion proteins constituted of elastin-like polypeptide (ELP), poly(aspartic acid) [poly(d)], and proteins-of-interest such as NanoLuc luciferase (Nluc) fused with replication initiation protein (Rep) (ELP-poly(d)-Nluc-Rep) or biotin acceptor peptide (BAP) (ELP-poly(d)-Nluc-BAP). Rep exhibited enzymatic conjugation activity with an optional DNA aptamer to protein nanoparticles. The transmembrane glycoprotein mucin 1 (MUC1)-binding DNA aptamer was conjugated to Rep as a model aptamer. Bioluminescence signals emitted from the Nluc domains were used to analyze the binding abilities. BAP contributed to binding to streptavidin-modified MPs via a biotin-streptavidin interaction. The MUC1-conjugated protein nanoparticles bound to MUC1-positive human breast cancer MCF-7 cells via MUC1 aptamers and streptavidin-conjugated MPs via BAP, leading to magnetic cell separation. The ratio of isolated MCF-7 cells via magnetic separation was 71.3% for the MCF-7 suspension at 1000 cells/1 mL. The MPs bound on recovered MCF-7 cells were removed by cooling at 4 °C to induce the dissociation of protein nanoparticles. Magnetic cell separation systems that use protein nanoparticles are a promising technology for biological research and diagnostic applications.