A microfluidic platform for real-time enumeration and high accuracy retrieval of a very low number of cells

In recent years, single cell isolation and analysis have become crucial, driven by the need to study rare cells in cell biology research, diagnostics, and personalized medicine. However, existing platforms for isolating small cell numbers are expensive, labor-intensive, and not widely accessible. To address this, we present a low-cost, repeatable microfluidic platform capable of retrieving 1-100 cells with high accuracy and minimal sample loss. The system utilizes a 2D hydrodynamic focusing chip and a pipette tip as a cell reservoir, enhanced by a flexible hydraulic reservoir (FHR) to prevent sample loss. Cells are collected using a syringe pump-driven flow, monitored in real-time under a microscope, and counted using image processing software. To validate the platform, MCF7 breast cancer cells were passed through the microchannel, with target retrieval numbers ranging from 1 to 100 cells. The average retrieved cell count was found to be 1.0 ± 0.0, 9.2 ± 2.4, 46.0 ± 5.9 and 98.5 ± 6.2 for 1, 10, 50, and 100 targeted number of cells, respectively. The counting accuracy of the code was demonstrated by the average deviation between the code count and retrieved number of cells being 0 ± 0.6, -0.3 ± 1.7, -1.6 ± 0.9, and 3.9 ± 4.8, respectively for 1, 10, 50, and 100 targeted cells. The process took less than 10 min, with cell counts matching targets closely and demonstrating high accuracy. Importantly, cell viability remained unaffected post-process. This method offers a cost-effective, robust solution for precise cell counting and retrieval, suitable for various downstream applications.