Microfluidic filter device with nylon mesh membranes efficiently dissociates cell aggregates and digested tissue into...

Single-cell analysis is a rapidly growing field used to explore cellular heterogeneity and detect rare cell populations. However, preparing high-quality single-cell suspensions remains a challenge. Current tissue dissociation methods often fail to fully break down tissues, leaving behind aggregates that reduce data accuracy and purity. Prior research has shown that mechanical and enzymatic dissociation techniques can be inconsistent, leading to variability in downstream results. This gap motivated the development of more efficient and reliable dissociation tools. No prior work had resolved the issue of simultaneous filtration and dissociation in a single device. Existing methods either remove aggregates or damage cells, but not both effectively. This limitation restricts the utility of single-cell analysis in clinical and research settings. The need for a scalable and gentle dissociation method is clear.

The study aimed to develop a microfluidic device that could both filter out large tissue fragments and dissociate smaller aggregates into single cells. The goal was to improve single-cell yield and purity while preserving cell viability. The researchers focused on creating a cost-effective and simple device that could be used in a variety of tissue types. They wanted to address the inefficiencies of current dissociation methods by integrating filtration and dissociation in one system. The study also aimed to test the device's effectiveness using cancer cell lines and murine tissues. The team sought to optimize pore sizes and flow rates to balance dissociation efficiency with cell damage prevention. They intended to validate the device using multiple tissue types to ensure broad applicability. The ultimate purpose was to provide a foundation for future integration into complete tissue analysis platforms.

The researchers designed a microfluidic device featuring two nylon mesh membranes with micron-sized pores. The first membrane operated under tangential flow to reduce clogging while filtering large tissue fragments. The second membrane dissociated smaller aggregates into single cells. The device was tested using cancer cell lines and murine tissues. Flow rates and pore sizes were varied to assess their impact on dissociation efficiency and cell damage. The first filtration step used larger pores to prevent clogging and allow tangential flow. The second filtration step used smaller pores to break down aggregates. The device was validated using minced and digested murine kidney, liver, and mammary tumor tissues. The performance was measured in terms of single-cell yield, purity, and viability.

The dual membrane microfluidic filter device increased single-cell numbers by at least 3-fold across multiple tissue types. In some cases, the yield improved by more than 10-fold. The device achieved these results without reducing cell viability. Using cancer cell lines, aggregates were effectively dissociated at high flow rates with pore sizes smaller than a single cell. However, pore sizes less than half the cell size caused significant cell damage. The researchers improved performance by using two filter devices in series. The second membrane's pore size was the primary determinant of single-cell yield and purity. The optimal combination of 50 and 15 μm membranes was identified for murine kidney tissue. The device was validated using liver and mammary tumor tissues, showing consistent performance across different tissue types.

The dual membrane microfluidic filter device effectively dissociates tissue aggregates into single cells while maintaining cell viability. The device's performance is primarily determined by the pore size of the second membrane. The combination of 50 and 15 μm membranes was found to be optimal for murine tissues. The device operates efficiently in minutes and does not require additional filtering before downstream applications. The results suggest that this device could improve single-cell analysis workflows by providing higher yield and purity. The study also proposes future integration of the device with other tissue processing technologies. The authors suggest that combining filtration and dissociation in one device is a promising approach for single-cell analysis. The device's simplicity and low cost make it suitable for broader adoption in research and clinical settings.