RESUMO
The characterization of individual cells within heterogeneous populations (e.g., rare tumor cells in healthy blood cells) has a great impact on biomedical research. To investigate the properties of these specific cells, such as genetic biomarkers and/or phenotypic characteristics, methods are often developed for isolating rare cells among a large number of background cells before studying their genetic makeup and others. Prior to using real-world samples, these methods are often evaluated and validated by spiking cells of interest (e.g., tumor cells) into a sample matrix (e.g., healthy blood) as model samples. However, spiking tumor cells at extremely low concentrations is challenging in a standard laboratory setting. People often circumvent the problem by diluting a solution of high-concentration cells, but the concentration becomes inaccurate after series dilution due to the fact that a cell suspension solution can be inhomogeneous, especially when the cell concentration is very low. We report on an alternative method for low-cost, accurate, and reproducible low-concentration cell spiking without the use of external pumping systems. By inducing a capillary force from sudden pressure drops, a small portion of the cellular membrane was aspirated into the reservoir tip, allowing for non-destructive single-cell transfer. We investigated the surface membrane tensions induced by cellular aspiration and studied a range of tip/tumor cell diameter combinations, ensuring that our method does not affect cell viability. In addition, we performed single-cell capture and transfer control experiments using human acute lymphoblastic leukemia cells (CCRF-CEM) to develop calibrated data for the general production of low-concentration samples. Finally, we performed affinity-based tumor cell isolation using this method to generate accurate concentrations ranging from 1 to 15 cells/mL.
RESUMO
The methods for isolating rare cells such as circulating tumor cells (CTCs) can be generally classified into two categories: those based on physical properties (e.g., size) and methods based on biological properties (e.g., immunoaffinity). CellSearch, the only FDA-approved method for the CTC-based cancer prognosis, relies on immunoaffinity interactions between CTCs and antibodies immobilized on magnetic particles. Immunoaffinity-based CTC isolation has also been employed in microfluidic devices, which show higher capture efficiency than CellSearch. We report here our investigation of combining size-based microfiltration into a microfluidic device with immunoaffinity for enhanced capture efficiency of CTCs. The device consists of four serpentine main channels, and each channel contains an array of lateral filters that create a two-dimensional flow. The main flow is through the serpentine channel, allowing the majority of the sample to pass by while the secondary flow goes through the lateral filters. The device design is optimized to make all fluid particles interact with filters. The filter sizes range from 24 to 12 µm, being slightly larger than or having similar dimension of CTCs. These filters are immobilized with antibodies specific to CTCs and thus they function as gates, allowing normal blood cells to pass by while forcing the interactions between CTCs and antibodies on the filter surfaces. The hydrodynamic force experienced by a CTC was also studied for optimal experimental conditions to ensure immunoaffinity-enabled cell capture. The device was evaluated by capturing two types of tumor cells spiked in healthy blood or a buffer, and we found that their capture efficiency was between 87.2 and 93.5%. The platform was further validated by isolating CTCs from blood samples of patients with metastatic pancreatic cancer.