Liposome-tethered supported lipid bilayer platform for capture and release of heterogeneous populations of circulating tumor cells.

Because of scarcity, vulnerability, and heterogeneity in the population of circulating tumor cells (CTCs), the CTC isolation system relying on immunoaffinity interaction exhibits inconsistent efficiencies for all types of cancers and even CTCs with different phenotypes in individuals. Moreover, releasing viable CTCs from an isolation system is of importance for molecular analysis and drug screening in precision medicine, which remains a challenge for current systems. In this work, a new CTC isolation microfluidic platform was developed and contains a coating of the antibody-conjugated liposome-tethered-supported lipid bilayer in a developed chaotic-mixing microfluidic system, referred to as the "LIPO-SLB" platform. The biocompatible, soft, laterally fluidic, and antifouling properties of the LIPO-SLB platform offer high CTC capture efficiency, viability, and selectivity. We successfully demonstrated the capability of the LIPO-SLB platform to recapitulate different cancer cell lines with different antigen expression levels. In addition, the captured CTCs in the LIPO-SLB platform can be detached by air foam to destabilize the physically assembled bilayer structures due to a large water/air interfacial area and strong surface tension. More importantly, the LIPO-SLB platform was constructed and used for the verification of clinical samples from 161 patients with different primary cancer types. The mean values of both single CTCs and CTC clusters correlated well with the cancer stages. Moreover, a considerable number of CTCs were isolated from patients' blood samples in the early/localized stages. The clinical validation demonstrated the enormous potential of the universal LIPO-SLB platform as a tool for prognostic and predictive purposes in precision medicine.

Multiomic characterization and drug testing establish circulating tumor cells as an ex vivo tool for personalized medicine.

Matching the treatment to an individual patient's tumor state can increase therapeutic efficacy and reduce tumor recurrence. Circulating tumor cells (CTCs) derived from solid tumors are promising subjects for theragnostic analysis. To analyze how CTCs represent tumor states, we established cell lines from CTCs, primary and metastatic tumors from a mouse model and provided phenotypic and multiomic analyses of these cells. CTCs and metastatic cells, but not primary tumor cells, shared stochastic mutations and similar hypomethylation levels at transcription start sites. CTCs and metastatic tumor cells shared a hybrid epithelial/mesenchymal transcriptome state with reduced adhesive and enhanced mobilization characteristics. We tested anti-cancer drugs on tumor cells from a metastatic breast cancer patient. CTC responses mirrored the impact of drugs on metastatic rather than primary tumors. Our multiomic and clinical anti-cancer drug response results reveal that CTCs resemble metastatic tumors and establish CTCs as an ex vivo tool for personalized medicine.

ViaChip for Size-based Enrichment of Viable Cells.

Live cells acquire different fates including apoptosis, necrosis, and senescence in response to stress and stimuli. Rapid and label-free enrichment of live cells from a mixture of cells adopting various cell fates remains a challenge. We developed a ViaChip for high-throughput enrichment of Viable cells via size-based separation on a multi-stage microfluidic Chip. Our chip takes advantage of the characteristic increase in cell size during cellular senescence and decreases during apoptosis and necrosis, in comparison to their viable and healthy counterparts. The core component of our ViaChip is a slanted and tunable 3D filter array in the vertical direction (z-gap) for rapid and continuous cell sieving. The shape of the 3D filter array is optimized for target cells to prevent clogging during continuous separation. We demonstrated enrichment of live human and mouse mesenchymal stem cells in culture and from live animals, as well as the removal of senescent and necrotic MSCs, respectively, achieving an enrichment efficiency of ~67% with the continuous flow at 1.5 mL/hour. With further improvements in throughput and separation efficiency, our ViaChip could find applications in cell-based drug screening for anti-cancer and anti-aging cell therapies.

Random and aligned electrospun PLGA nanofibers embedded in microfluidic chips for cancer cell isolation and integration with air foam technology for cell release.

BACKGROUND: Circulating tumor cells (CTCs) comprise the high metastatic potential population of cancer cells in the blood circulation of humans; they have become the established biomarkers for cancer diagnosis, individualized cancer therapy, and cancer development. Technologies for the isolation and recovery of CTCs can be powerful cancer diagnostic tools for liquid biopsies, allowing the identification of malignancies and guiding cancer treatments for precision medicine. METHODS: We have used an electrospinning process to prepare poly(lactic-co-glycolic acid) (PLGA) nanofibrous arrays in random or aligned orientations on glass slips. We then fabricated poly(methyl methacrylate) (PMMA)-based microfluidic chips embedding the PLGA nanofiber arrays and modified their surfaces through sequential coating with using biotin-(PEG)7-amine through EDC/NHS activation, streptavidin (SA), and biotinylated epithelial-cell adhesion-molecule antibody (biotin-anti-EpCAM) to achieve highly efficient CTC capture. When combined with an air foam technology that induced a high shear stress and, thereby, nondestructive release of the captured cells from the PLGA surfaces, the proposed device system operated with a high cell recovery rate. RESULTS: The morphologies and average diameters of the electrospun PLGA nanofibers were characterized using scanning electron microscopy (SEM) and confocal Raman imaging. The surface chemistry of the PLGA nanofibers conjugated with the biotin-(PEG)7-amine was confirmed through time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging. The chip system was studied for the effects of the surface modification density of biotin-(PEG)7-amine, the flow rates, and the diameters of the PLGA nanofibers on the capture efficiency of EpCAM-positive HCT116 cells from the spiked liquid samples. To assess their CTC capture efficiencies in whole blood samples, the aligned and random PLGA nanofiber arrays were tested for their abilities to capture HCT116 cells, providing cancer cell capture efficiencies of 66 and 80%, respectively. With the continuous injection of air foam into the microfluidic devices, the cell release efficiency on the aligned PLGA fibers was 74% (recovery rate: 49%), while it was 90% (recovery rate: 73%) on the random PLGA fibers, from tests of 200 spiked cells in 2 mL of whole blood from healthy individuals. Our study suggests that integrated PMMA microfluidic chips embedding random PLGA nanofiber arrays may be suitable devices for the efficient capture and recovery of CTCs from whole blood samples.

Scalable Multilayer Cell Collector to Capture Circulating Tumor Cells with an Unlimited Volume Capacity.

Circulating tumor cells (CTCs) have been suggested as the precursors of metastatic cancer. CTC-based characterization has thus been used to monitor tumor status before the onset of metastasis and has shown to be an independent factor. The low abundance of CTCs, however, makes it challenging to employ CTC as a clinical routine, thus making it impossible to address tumor heterogeneity. Here, we present a cell collection prototype for an efficient capture of CTCs from a large volume of body fluids such as blood. An antibody-PEG modified multilayer matrix column is engineered and connected to an apheresis-based circulation system. This setup allows us to capture CTCs repetitively from an unlimited sample volume through the circulation system, thereby increasing the capture count. Compared to conventional CTC capturing devices where the sample handling is generally limited to 1-10 mL, our collector is able to handle a wide range of fluidic sample (40-2000 mL) at a high flow rate (400 mL/min). By processing 90 min in circulation, we obtained an average capture efficiency of at least 75% for the colorectal cancer cell line HCT116 spiked in either 40-200 mL of buffer solution or 40 mL of a whole blood sample. This result highlights a possibility to construct personalized CTC libraries through high-throughput CTC collection for the study of tumor heterogeneity in precision medicine.

Promoting Multivalent Antibody-Antigen Interactions by Tethering Antibody Molecules on a PEGylated Dendrimer-Supported Lipid Bilayer.

To efficiently isolate maximal quantity of circulating tumor cells (CTCs) and circulating tumor cell microembolis (CTMs) from patient blood by antibody coated microfluidics, a multifunctional, pegylated polyamidoamine-dendrimers conjugated supported lipid bilayer surface construct was proposed to enhance accessibility of antibody molecules to the antigen molecules on target CTCs. The combination of a hydrated, stretchable dendrimer and a laterally mobile supported lipid bilayer (SLB) provide attached antibody molecules with 2.5-dimensional chain movement, achieving multivalency between the surface antibody and cell antigen molecules. An over 170% enhancement is distinctive for Panc-1 cells that expresses low antigen level. Of seven pancreatic ductal adenocarcinoma patients, an average 440 single CTCs and 90 CTMs were collected in 2 mL of peripheral blood, which were 1.6 times and 2.3 times more, than those captured by the SLB-only microfluidics. In summary, we have demonstrated a material design to enhance multivalent antibody-antigen interaction, which is useful for rare cell enrichment and cancer detection.

Nonfouling hydrophilic poly(ethylene glycol) engraftment strategy for PDMS/SU-8 heterogeneous microfluidic devices.

We report a novel nonfouling passivation method using poly(ethylene glycol) (PEG) engraftment on the surfaces of poly(dimethylsiloxane) (PDMS) microfluidic devices sealed with SU-8. To achieve bonding between the PDMS and SU-8 surfaces, the PDMS surface was first functionalized with amines by treatment with 3-aminopropyltrimethoxysilane (APTMS) for subsequent reaction with epoxide functional groups on SU-8 surfaces. To modify the heterogeneous surfaces of the resulting PDMS/SU-8 microfluidic device further, the remaining SU-8 surfaces were amino functionalized using ethylene diamine (EDA), followed by treating both amino-functionalized PDMS and SU-8 surfaces with mPEG-NHS (N-hydroxysuccinimide) through an amine-NHS reaction for facile PEG immobilizations, thus simultaneously modifying both PDMS and SU-8 surfaces in one reaction. Detailed surface analyses such as the water contact angle, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM) were conducted to confirm the chemical reactions and characterize the resulting surface properties. To test the efficacy of this surface-modification strategy, we conducted nonspecific protein and particle binding tests using microfluidic devices with and without modifications. The PEG-modified PDMS/SU-8 device surfaces showed a 64.5% reduction in nonspecific bovine serum albumin (BSA) adsorption in comparison to that of the unmodified surfaces and 92.0 and 95.8% reductions in microbead adhesion under both stagnant and flowing conditions, respectively.

Nonbiofouling polymer brush with latent aldehyde functionality as a template for protein micropatterning.

A novel, nonfouling polymer brush, poly-N-[(2,3-dihydroxypropyl)acrylamide] (PDHPA), containing latent aldehyde groups, was synthesized by surface initiated atom transfer radical polymerization (SI-ATRP). The synthetic parameters were adjusted to produce brushes with varying graft densities and molecular weights. High-density PDHPA brushes successfully prevented the nonspecific protein adsorption from single protein solutions as well as from human platelet poor plasma. Patterns of nonfouling PDHPA and reactive PDHPA-aldehyde domains on the brush surface were created by a combination of photo and wet chemical lithography from a single homogeneous PDHPA brush. Successful micropatterning of single proteins and multiple proteins were achieved using this novel substrate. The high-density brush prevented the diffusion of large proteins into the brush, while a monolayer of covalently coupled proteins was formed on the PDHPA-aldehyde domains. Atomic force microscopy (AFM) force measurements using a biotin coupled AFM tip showed that covalently coupled streptavidin retained its activity, while PDHPA domains showed little nonspecific adsorption of streptavidin. The current study avoids tedious and complicated synthetic processes employed in conventional approaches by providing a novel approach to protein micropatterning from a single, multifunctional polymer brush.

An investigation of vibration-induced protein desorption mechanism using a micromachined membrane and PZT plate.

A micromachined vibrating membrane is used to remove adsorbed proteins on a surface. A lead zirconate titanate (PZT) composite (3 x 1 x 0.5 mm) is attached to a silicon membrane (2,000 x 500 x 3 microm) and vibrates in a flexural plate wave (FPW) mode with wavelength of 4,000/3 microm at a resonant frequency of 308 kHz. The surface charge on the membrane and fluid shear stress contribute in minimizing the protein adsorption on the SiO(2) surface. In vitro characterization shows that 57 +/- 10% of the adsorbed bovine serum albumin (BSA), 47 +/- 13% of the immunoglobulin G (IgG), and 55.3~59.2 +/- 8% of the proteins from blood plasma are effectively removed from the vibrating surface. A simulation study of the vibration-frequency spectrum and vibrating amplitude distribution matches well with the experimental data. Potentially, a microelectromechanical system (MEMS)-based vibrating membrane could be the tool to minimize biofouling of in vivo MEMS devices.

Self-assembled monothiol-terminated hyperbranched polyglycerols on a gold surface: a comparative study on the structure, morphology, and protein adsorption characteristics with linear poly(ethylene glycol)s.

Monothiol-terminated hyperbranched polyglycerols (HPGs) were synthesized by ring-opening polymerization of glycidol from partially deprotonated 2,2'-dihydroxyethane disulfide as the initiator and subsequent reduction of the disulfide group. Two molecular weights of HPG thiols were synthesized. The molecular weights of the polymers were determined by MALDI-TOF analysis, and the presence of thiol was verified by Ellman's assay. The self-assembly of HPG thiols on gold was studied and compared with that of linear poly(ethylene glycol) (PEG) thiols utilizing various surface analysis techniques. Monothiol-functionalized HPGs readily adsorbed to a gold surface and formed highly uniform thin films on the surface. The graft density of the HPG layer decreased with an increase in the molecular weight of the polymer. The amount of polymer on the surface increased with increasing incubation concentration and saturated above 6 g/L polymer concentration. Generally, HPG thiols gave lower graft density compared to linear PEG thiols of similar molecular weight. AFM morphological studies showed that HPG thiols form more uniform and smooth surface films compared to PEG thiols. Incubation of a polymer-coated surface (HPG thiols and PEG thiols) with bovine serum albumin and immunoglobulin showed that the high molecular weight hyperbranched polyglycerol was more resistant to protein adsorption than linear PEG of similar molecular weight or lower molecular weight HPG. The protein adsorption decreased with increasing graft density of the HPG chains on the surface. Our results show that HPG could be a good alternative to PEG in the development of nonfouling functional surfaces.

Electric field and vibration-assisted nanomolecule desorption and anti-biofouling for biosensor applications.

A novel anti-fouling mechanism based on the combined effects of electric field and shear stress is reported. A lead zirconate titanate (PZT) composite is used to generate an electric field and an acoustic streaming shear stress that increase nanomolecule desorption. In vitro characterization showed that (1) 58+/-5.5% and 39+/-5.2% of adsorbed bovine serum albumin (BSA) proteins can be effectively removed from fired silver and titanium coated PZT plate, respectively; and (2) 43+/-9.7% of the anti-mouse immunoglobulin G (IgG) can be effectively removed from a fired silver coated PZT plate. Theoretical calculations on protein-surface interactions (van der Waals (VDW), electrostatic, and hydrophobic) and shear stress describe the mechanism for protein desorption from model surfaces. We have shown that the applied electric potential is the major contributor in reducing the adhesive force between protein and surface, and the desorbed protein is taken away by acoustic streaming shear stress. We strongly believe that the present method offers the possibility of minimizing nanomolecule adsorption without further surface treatment.