Selective Cell Capture Research Articles

Fe0-MAP Prepared Glycosurfaces for Selective Cell Capture: From Adherent to Suspended Cells.

The specific capture of live cells is crucial for various biomedical applications. Existing methods often are limited by complex production processes. This study introduces Fe0-mediated monomer-adaptation polymerization (Fe0-MAP), a convenient and rapid synthesis approach for selective cell capture using surface-engineered glycopolymer brushes. This method utilizes surface-initiated zerovalent iron-mediated reversible-deactivation radical polymerization (Fe0-SI-RDRP), offering advantages like simplicity, biocompatibility and oxygen-tolerance due to the use of iron sheet as catalysts. We successfully employed Fe0-MAP to selective capture both adherent (HeLa, L929) and suspended cells (Ramos, U937) in mammalian cell cultures. Combining excellent biocompatibility, specific and reusable cell capture capabilities, and applicability to suspended cells, Fe0-MAP establishes itself as a promising strategy for selective cell capture, holding significant potential for diverse biomedical applications.

Light-mediated protein functionalization of photoclickable hydrogel interface for selective cell capture and dot blotting assay

The construction of versatile functional hydrogel interfaces holds promising prospects in biosensing and bioengineering. Herein, we introduced a light-induced protein conjugation strategy for on-demand surface modification of hydrogel interface based on the photoclick cyclization between primary amine and o-nitrobenzyl alcohol. We achieved the on-demand protein conjugation by grafting the molecular plugin, 4-(hydroxymethyl)-3-nitrobenzoic acid (HNBA), onto the hydrogel surface, followed by the mask-aided photoclick reaction with the protein of interest. This method enables the creation of protein patterns on hydrogel interface with a lower limit of pattern width at ∼70 μm. With this method, we demonstrated the surface engineering of epidermal growth factor (EGF) on hydrogel interface for selective capture of EGF receptor-positive cancer cells with an efficiency over 80%. Moreover, we applied the mask-aided photoclick conjugation method for antigen capture and developed a photoclickable hydrogel interface-based dot blotting assay. Due to the high-efficient antigen capture of photoclick conjugation, the photoclickable hydrogel interface-based dot blotting assay shows improved sensitivity for antigen detection with a limit of detection as 0.065 ng. We believed that this light-induced protein conjugation method holds the potential as a robust strategy for the construction of bioactive hydrogel interfaces for various bio-related applications.

Optimizing Immunofunctionalization and Cell Capture on Micromolded Hydrogels via Controlled Oxygen-Inhibited Photopolymerization.

With circulating tumor cells (CTCs) playing a critical role in cancer metastasis, the quantitation and characterization of CTCs promise to provide precise diagnostic and prognostic information in service of personalized therapies. However, as CTCs are extremely rare, high yield, high purity strategies are required to target and isolate CTCs from patient samples. Recently, we demonstrated the selective capture of CTCs upon antibody-functionalized polyethylene glycol diacrylate (PEGDA) hydrogels photopolymerized within polydimethylsiloxane (PDMS) microfluidic molds. Isolated CTC purity was subsequently enriched by selectively releasing desired cells from photodegradable hydrogel capture surfaces. However, the fabrication of these acrylate-based hydrogels by photopolymerization is subject to oxygen inhibition, which dramatically affects the physical and chemical properties of hydrogel interfaces formed in proximity to PDMS boundaries. To evaluate how antibody conjugation density and cell capture is impacted by fabrication parameters affected by oxygen inhibition, PEGDA hydrogel features were polymerized within PDMS micromolds under different UV exposure conditions and linker (acrylate-PEG-biotin) concentrations. Predictions of acrylate conversion throughout the hydrogel feature were performed using a 1D reaction-diffusion model that describes oxygen-inhibited photopolymerization. The functional consequences of photopolymerization parameters and solution stoichiometry on CTC capture were experimentally quantified and evaluated. Results show that hydrogel surfaces polymerized under shorter exposure times and with higher linker concentrations display superior functionalization and higher CTC capture efficiency. Conversely, highly cross-linked hydrogel surfaces polymerized under longer exposure times are insensitive to functionalization and display poor capture, regardless of linker concentration. By highlighting the importance of oxygen-inhibited photopolymerization, these findings provide guidelines to design micromolded hydrogels with controlled ligand expression. In addition to enhancing the selective cell capture capacity of immunofunctional hydrogels, the ability to quantifiably design hydrogel interfaces described here will improve the sensitivity of hydrogel biosensors, provide a platform to finely screen cell-matrix interactions, and generally enhance the fidelity of micromolded hydrogel features.

Impact of REDV peptide density and its linker structure on the capture, movement, and adhesion of flowing endothelial progenitor cells in microfluidic devices

Ligand-immobilization to stents and vascular grafts is expected to promote endothelialization by capturing flowing endothelial progenitor cells (EPCs). However, the optimized ligand density and linker structure have not been fully elucidated. Here, we report that flowing EPCs were selectively captured by the REDV peptide conjugated with a short linker. The microchannel surface was modified with the REDV peptide via Gly-Gly-Gly (G3), (Gly-Gly-Gly)3 (G9), and diethylene glycol (diEG) linkers, and the moving velocity and captured ratio were evaluated. On the unmodified microchannels, the moving velocity of the cells exhibited a unimodal distribution similar to the liquid flow. The velocity of the endothelial cells and EPCs on the peptide-immobilized surface indicated a bimodal distribution, and approximately 20 to 30% of cells moved slower than the liquid flow, suggesting that the cells were captured and rolled on the surface. When the immobilized ligand density was lower than 1 molecule/nm2, selective cell capture was observed only in REDV with G3 and diEG linkers, but not in G9 linkers. An in silico study revealed that the G9 linker tends to form a bent structure, and the REDV peptide is oriented to the substrate side. These results indicated that REDV captured the flowing EPC in a sequence-specific manner, and that the short linker was more adequate.

A superior dead cell removal platform using Akadeum’s BACS microbubbles

Abstract Single cell sequencing (SCS) is one of the most commonly used analytical techniques for cellular characterization and has become a mainstay in immune profiling. As SCS has gained in popularity and improved in sensitivity, it has become apparent that ambient nucleic acids can have a major impact on the quality and reproducibility of obtained SCS results. Since the bulk of ambient nucleic acids in a sample come from dead or dying cells that have lost membrane integrity, one way to dramatically reduce ambient nucleic acids and decrease background noise in SCS is to remove dead cells from the sample prior to sequencing. To address the challenge presented by contaminating dead cells, Akadeum has developed a Buoyancy Activated Cell Sorting (BACS™) microbubble approach for depleting dead cells from biological samples. Leveraging the power of gravity to drive self-isolation, Akadeum’s BACS microbubbles represent some of the gentlest isolations available; therefore, they are ideally suited for applications where cell viability is integral to success of the experiment. Additionally, BACS microbubbles from Akadeum are quick and easy to use since they require only minimal manipulation and do not rely on any extra equipment or consumables. Depletion of dead cells is achieved through the selective capture of cells with exposed phosphatidylserine with Annexin V conjugated BACS microbubbles. Once mixed with the sample, the Annexin V BACS microbubbles capture dead cells and float them to the sample surface for removal leaving behind healthy and untouched cells. Samples exposed to the Annexin V BACS microbubbles demonstrated significantly reduced dead cell populations and ultimately yielded more reliable results.

Ultra-low fouling photocrosslinked coatings for the selective capture of cells expressing CD44

The effective control of biointerfacial interactions is of outstanding interest in a broad range of biomedical applications, ranging from cell culture tools to biosensors and implantable medical devices. For many of these applications, highly specific interactions between cells and material surfaces are desired. Sophisticated control over these interactions requires reducing or preventing non-specific interactions on the one hand and displaying highly specific signals that can be recognized by extracellular receptors on the other. We have recently developed ultra-low fouling coatings that can be applied in a single step using photoreactive copolymers of 2-hydroxypropyl acrylamide and N-benzophenone acrylamide. Here, we have expanded this approach by incorporating polymerizable peptide monomers into these copolymers. The monomers QQGWFGAGK(acrylamide) and acrylamide-GAGQQGWF were synthesized after identifying the QQGWF sequence as a binding motif for CD44 by phage display for the first time. Our results demonstrate that UV-crosslinked coatings fabricated using the QQGWFGAGK(acrylamide) monomer are effective at selectively binding hMSC in the presence of HepG2 and HEK293 cells due to the difference in CD44 expression. Our results also demonstrate that the peptide modified coatings retain their low biofouling character using a BCA protein binding assay as well as an E. coli bacterial attachment assay over a 24 h period. Our approach provides an alternative to traditional integrin-mediated selective cell binding on surfaces and opens the door to new diagnostic applications, exploiting the fact that the transmembrane protein CD44 is highly expressed in multiple diseases.

Imaging-assisted hydrogel formation for single cell isolation

We report a flexible single-cell isolation method by imaging-assisted hydrogel formation. Our approach consists of imaging-aided selective capture of cells of interest by encasing them into a polymeric hydrogel, followed by removal of unwanted cells and subsequent release of isolated cells by enzymatic hydrogel degradation, thus offering an opportunity for further analysis or cultivation of selected cells. We achieved high sorting efficiency and observed excellent viability rates (>98%) for NIH/3T3 fibroblasts and A549 carcinoma cells isolated using this procedure. The method presented here offers a mask-free, cost-efficient and easy-to-use alternative to many currently existing surface-based cell-sorting techniques, and has the potential to impact the field of cell culturing and isolation, e.g. single cell genomics and proteomics, investigation of cellular heterogeneity and isolation of best performing mutants for developing new cell lines.

Affinity-Enhanced CTC-Capturing Hydrogel Microparticles Fabricated by Degassed Mold Lithography.

Technologies for the detection and isolation of circulating tumor cells (CTCs) are essential in liquid biopsy, a minimally invasive technique for early diagnosis and medical intervention in cancer patients. A promising method for CTC capture, using an affinity-based approach, is the use of functionalized hydrogel microparticles (MP), which have the advantages of water-like reactivity, biologically compatible materials, and synergy with various analysis platforms. In this paper, we demonstrate the feasibility of CTC capture by hydrogel particles synthesized using a novel method called degassed mold lithography (DML). This technique increases the porosity and functionality of the MPs for effective conjugation with antibodies. Qualitative fluorescence analysis demonstrates that DML produces superior uniformity, integrity, and functionality of the MPs, as compared to conventional stop flow lithography (SFL). Analysis of the fluorescence intensity from porosity-controlled MPs by each reaction step of antibody conjugation elucidates that more antibodies are loaded when the particles are more porous. The feasibility of selective cell capture is demonstrated using breast cancer cell lines. In conclusion, using DML for the synthesis of porous MPs offers a powerful method for improving the cell affinity of the antibody-conjugated MPs.

Antibody-coated microstructures for selective isolation of immune cells in blood.

Cell isolation from blood is an important process for diagnosing immune diseases. There are still demands for a user-friendly approach to achieve high cell extraction efficiency and purity of a target immune cell subtype for more promising diagnosis and monitoring. For selective immune cell isolation, we developed a microstructured device, which consists of antibody-coated micropillars and micro-sieve arrays, for isolating a target immune cell subtype from bovine blood samples. The focusing micropillars can guide immune cells flowing to the subsequent micro-sieves based on deterministic lateral shifts of the cells. The arrangement of these microstructures is characterized and configured for the maximal cell capture rate. Surface modification with a selected antibody offers selective cell capture in the micro-sieves based on the antigen-antibody reaction. We prepare a cell mixture of human CD14-expressing leukemia cells (THP-1) and epithelial cells (MDA-MB-231) in diluted blood to characterize the cell isolation operation, with a selective cell isolation yield of >80%, cell purity of ∼100% and cell viability of >93%. Together, this microstructured device strategy can achieve high-yield selective isolation of immune cells from blood samples and support downstream genetic and biochemical cell analyses, contributing to the medical diagnosis of a broad range of immune diseases.

Surfaces that Adhesively Discriminate Breast Epithelial Cell Lines and Lymphocytes in Buffer and Human Breast Milk.

We report new surface coatings that adhesively distinguish three breast epithelial cell lines (MCF-10A, MCF-7, and TMX2-28) when cell suspensions in buffer or breast milk are flowed over the coatings. We also report the selective capture of epithelial cells and rejection of Jurkat lymphocytes, with average selectivities exceeding 60 and captured cell purities often exceeding >99%. The surfaces achieve the dual goals of selective cell capture and resistance to fouling by proteins and other components of breast milk. The coatings do not rely on antibody targeting of cell surface markers but instead contain polycation chains embedded within a layer of end-tethered poly(ethylene glycol) (PEG) chains. The PEG, somewhat shielding the polycations, prevents surface fouling by proteins, nondesired cells, and other milk components, while the polycations produce electrostatic attractions that are heterogeneous on nanoscopic length scales. These electrostatic heterogeneities on the engineered coating, shown to produce curvature-selective particle capture in other studies, produce cell selectivity here. The ability of the engineered surfaces to discriminate these cell lines via an electrostatic driving force is remarkable, as the cells are of very similar surface charge as evidenced by their nearly identical ζ-potentials. The current surfaces, which likely distinguish cells based on their electrostatic surface landscape combined with other factors, adhesively distinguish cell lines that may differ only slightly in their expression of a surface marker, or cancer cells that minimally express EpCAM but which have different distributions of electrostatic charge on their surfaces. These surfaces are among the first to be documented for the compatibility of a polymer brush with human breast milk and may find use in technologies that capture cells from human breast milk or other complex fluids for cancer risk assessment.

One-step fabrication of flexible nanotextured PDMS as a substrate for selective cell capture

Efficient cancer cell capture has been previously demonstrated on functionalized surfaces with defined nanotopography such as nanoscale texture. However, conventional additive and subtractive methods to achieve nanotexture require access to specialized nanofabrication equipment within a dedicated cleanroom environment. Here, we present a technique to create flexible polydimethylsiloxane (PDMS) surfaces with high roughness (Rq ∼ 680 nm) using a molding approach that can be performed in a standard laboratory environment. We also demonstrate a one-step integration of nanotextured PDMS into a reversibly sealed easy access modular microfluidic platform to simplify cell capture workflows (e.g. cell introduction, capture, and isolation). In our proof of concept, we characterize nanotextured PDMS surfaces and investigate the effect of increased surface area on cancer cell adhesion strength and capture compared to non-textured (plain) PDMS. We found that cells attached more strongly to antibody-functionalized nanotextured surfaces (26% ± 5% increase in threshold fluid shear stress, τ50%) and captured 71% ± 19% more cancer cells than functionalized plain surfaces. Our reversibly sealed microfluidic platform enabled a user-friendly method to access cells for post-capture analysis, and we report an efficient nucleic acid isolation process. We anticipate the easy fabrication, one-step microfluidic integration, and streamlined experimental workflow will simplify the incorporation of nanotextured substrates in applications that investigate cell–surface interactions.

解離型分子を介した抗体固定化グラフト表面を用いた選択的な細胞捕獲・回収による細胞分離

解離型分子を介した抗体固定化グラフト表面を用いた選択的な細胞捕獲・回収による細胞分離

Catch-and-Release of Target Cells Using Aptamer-Conjugated Electroactive Zwitterionic Oligopeptide SAM

Nucleic acid aptamers possess attractive features such as specific molecular recognition, high-affinity binding, and rapid acquisition and replication, which could be feasible components for separating specific cells from other cell types. This study demonstrates that aptamers conjugated to an oligopeptide self-assembled monolayer (SAM) can be used to selectively trap human hepatic cancer cells from cell mixtures containing normal human hepatocytes or human fibroblasts. Molecular dynamics calculations have been performed to understand how the configurations of the aptamers are related to the experimental results of selective cell capture. We further demonstrate that the captured hepatic cancer cells can be detached and collected along with electrochemical desorption of the oligopeptide SAM, and by repeating these catch-and-release processes, target cells can be enriched. This combination of capture with aptamers and detachment with electrochemical reactions is a promising tool in various research fields ranging from basic cancer research to tissue engineering applications.

DNA-SMART: Biopatterned Polymer Film Microchannels for Selective Immobilization of Proteins and Cells.

A novel SMART module, dubbed "DNA-SMART" (DNA substrate modification and replication by thermoforming) is reported, where polymer films are premodified with single-stranded DNA capture strands, microthermoformed into 3D structures, and postmodified with complementary DNA-protein conjugates to realize complex biologically active surfaces within microfluidic devices. As a proof of feasibility, it is demonstrated that microchannels presenting three different proteins on their inner curvilinear surface can be used for selective capture of cells under flow conditions.

Selective Adhesive Cell Capture without Molecular Specificity: New Surfaces Exploiting Nanoscopic Polycationic Features as Discrete Adhesive Units.

This work explored how molecularly non-specific polycationic nanoscale features on a collecting surface control kinetic and selectivity aspects of mammalian cell capture. Key principles for selective collector design were demonstrated by comparing the capture of two closely related breast cancer cell lines: MCF-7 and TMX2-28. TMX2-28 is a tamoxifen-selected clone of MCF-7. The collector was a silica surface, negatively-charged at pH 7.4, containing isolated molecules (~ 8 nm diameter) of the cationic polymer, poly(dimethyl-aminoethylmethacrylate), pDMAEMA. Important in this work is the non-selective nature of the pDMAEMA interactions with cells: pDMAEMA generally adheres negatively charged particles and cells in solution. We show here that selectivity towards cells results from collector design: this includes competition between repulsive interactions involving the negative silica and attractions to the immobilized pDMAEMA molecules, the random pDMAEMA arrangement on the surface, and the concentration of positive charge in the vicinity of the adsorbed pDMAEMA chains. The latter act as nanoscopic cationic surface patches, each weakly attracted to negatively-charged cells. Collecting surfaces engineered with an appropriate amount pDMAEMA, exposed to mixtures of MCF-7 and TMX2-28 cells preferentially captured TMX2-28 with a selectivity of 2.5. (This means that the ratio of TMX2-28 to MCF cells on the surface was 2.5 times their compositional ratio in free solution.) The ionic strength-dependence of cell capture was shown to be similar to that of silica microparticles on the same surfaces. This suggests that the mechanism of selective cell capture involves nanoscopic differences in the contact areas of the cells with the collector, allowing discrimination of closely related cell line-based small scale features of the cell surface. This work demonstrated that even without molecular specificity, selectivity for physical cell attributes produces adhesive discrimination.

High-efficiency single cell encapsulation and size selective capture of cells in picoliter droplets based on hydrodynamic micro-vortices.

Single cell analysis has emerged as a paradigm shift in cell biology to understand the heterogeneity of individual cells in a clone for pathological interrogation. Microfluidic droplet technology is a compelling platform to perform single cell analysis by encapsulating single cells inside picoliter-nanoliter (pL-nL) volume droplets. However, one of the primary challenges for droplet based single cell assays is single cell encapsulation in droplets, currently achieved either randomly, dictated by Poisson statistics, or by hydrodynamic techniques. In this paper, we present an interfacial hydrodynamic technique which initially traps the cells in micro-vortices, and later releases them one-to-one into the droplets, controlled by the width of the outer streamline that separates the vortex from the flow through the streaming passage adjacent to the aqueous-oil interface (dgap). One-to-one encapsulation is achieved at a dgap equal to the radius of the cell, whereas complete trapping of the cells is realized at a dgap smaller than the radius of the cell. The unique feature of this technique is that it can perform 1. high efficiency single cell encapsulations and 2. size-selective capturing of cells, at low cell loading densities. Here we demonstrate these two capabilities with a 50% single cell encapsulation efficiency and size selective separation of platelets, RBCs and WBCs from a 10× diluted blood sample (WBC capture efficiency at 70%). The results suggest a passive, hydrodynamic micro-vortex based technique capable of performing high-efficiency single cell encapsulation for cell based assays.

Development of a selective cell capture and release assay: impact of clustered RGD ligands.

There is a growing interest in isolating tumor cells from biological samples. Considering that circulating tumor cells can be rare in blood, it appears challenging to capture these cells onto a surface with high selectivity and sensitivity. In the present paper, we describe the design of functionalized surfaces aimed at selectively capturing tumor cells by using an RGD peptide ligand with either a tetrameric or a monomeric presentation. β-Cyclodextrin-coated self-assembled monolayers were used as platforms for the binding of RGD ligands endowed with a redox ferrocene cluster. The dissociation of the inclusion complex on the surface accounts for the release of the captured cells upon the electrochemical oxidation of ferrocene. For this purpose, we determined suitable RGD densities for both monovalent and tetravalent ligand presentations. The results indicate that the clustered RGD architecture efficiently improves selective cell capture at a very low RGD surface density (∼10 RGD per μm2) compared to the monovalent presentation (∼1000 RGD per μm2).

Multiplexed labeling system for high-throughput cell sorting

Flow cytometry and fluorescence activated cell sorting techniques were designed to realize configurable classification and separation of target cells. A number of cell phenotypes with different functionalities have recently been revealed. Before simultaneous selective capture of cells, it is desirable to label different samples with the corresponding dyes in a multiplexing manner to allow for a single analysis. However, few methods to obtain multiple fluorescent colors for various cell types have been developed. Even when restricted laser sources are employed, a small number of color codes can be expressed simultaneously. In this study, we demonstrate the ability to manifest DNA nanostructure-based multifluorescent colors formed by a complex of dyes. Highly precise self-assembly of fluorescent dye-conjugated oligonucleotides gives anisotropic DNA nanostructures, Y- and tree-shaped DNA (Y-DNA and T-DNA, respectively), which may be used as platforms for fluorescent codes. As a proof of concept, we have demonstrated seven different fluorescent codes with only two different fluorescent dyes using T-DNA. This method provides maximum efficiency for current flow cytometry. We are confident that this system will provide highly efficient multiplexed fluorescent detection for bioanalysis compared with one-to-one fluorescent correspondence for specific marker detection.

A supramolecular pyrenyl glycoside-coated 2D MoS2 composite electrode for selective cell capture

Here we demonstrate the simple construction and characterization of a pyrenyl glycoside-coated 2D MoS2 material composite capable of selectively capturing proteins and live cells on an electrode, as determined by differential pulse voltammetry.

Antibody modified porous silicon microparticles for the selective capture of cells.

Herein, the ability of porous silicon (PSi) particles for selectively binding to specific cells is investigated. PSi microparticles with a high reflectance band in the reflectivity profile are fabricated, and subsequently passivated and modified with antibodies via the Cu(I)-catalyzed alkyne-azide cycloaddition reaction and succimidyl activation. To demonstrate the ability of the antibody-modified PSi particles to selectively bind to one cell type over others, HeLa cells were transfected with surface epitopes fused to fluorescent proteins. The antibody-functionalized PSi particles showed good selectivity for the corresponding surface protein on HeLa cells, with no significant cross-reactivity. The results are important for the application of PSi particles in cell sensing and drug delivery.