Extraction of Material Properties for Optimised Acoustophoresis-Based Particle and Cell Separation Using Flow Cytometry Data

Abstract

Acoustophoresis enables label-free separation of particles and cells in microfluidic sys- tems, but systematic methods for analysing and comparing separation behaviour re- main limited. In this thesis, a workflow based on the mobility ratio was evaluated as an experimental framework for characterising acoustophoretic separations, using flow cytometry as the analytical tool.

Separation experiments were performed on polystyrene beads and human cell lines (K562 and Jurkat) under varying separation voltages and buffer conditions. Sepa- ration quality was quantified using flow cytometry, and mobility ratios were experi- mentally derived from the voltages corresponding to equal outlet splitting (SQ50) of the separated particles and cells.

For polystyrene beads, the measured mobility ratios showed good agreement with the- oretical expectations based on particle size, demonstrating robustness of the SQ50- method. The experiments with cells exhibited increased variability, both in the mea- sured and expected values, reflecting the heterogeneity of biological samples. While the measured mobility ranges were within the expected range, the SQ50-points showed greater variation between runs compared to polystyrene beads. This indicates that the method is less robust for cells, however it can still be applied. Addition of 15% iodix- anol did not greatly affect the acoustic behaviour of K562 and Jurkat cells. Separation of K562 and Jurkat cells resulted in mobility ratios close to unity, consistent with their similar acoustic properties and overlapping size distributions.

Together, these findings demonstrate that the mobility ratio is a robust and exper- imentally accessible parameter for characterising acoustophoretic separations, while also clarifying its limits for biological samples.

Popular Abstract

Using sound to sort cells more efficiently

Separating cells is a key-step in medical research and diagnostics, but today's methods often require time-consuming trial and error. This thesis explores how sound waves and a simple comparison metric can make cell separation faster, more predictable and easier to optimise.

Separating cells and particles is a fundamental task in biomedical research, for example when analysing blood samples or preparing cells for disease diagnostics. One promising technique is acoustophoresis, which uses ultrasonic sound waves inside tiny microfluidic channels to move particles and cells based on their physical properties. Unlike many conventional methods, acoustophoresis can separate cells without chemical labels, preserving cell integrity.

Despite its advantages, acoustophoresis is often difficult to optimise. Several experimental parameters, such as flow rates and applied voltage, must be carefully tuned, and this is usually done by manually testing many combinations. This process is time-consuming and requires expert knowledge, which limits the technique’s wider use.

In this thesis, a workflow based on the mobility ratio was evaluated as a way to simplify and systematise acoustophoretic separations. The mobility ratio is a relative measure that compares how strongly two types of particles or cells respond to sound waves. By measuring the voltage at which two species are split equally between outlets, the mobility ratio can be experimentally determined and used to predict separation performance.

The method was tested using polystyrene beads of different sizes as well as two human cell lines, K562 and Jurkat cells. Flow cytometry was used to analyse the separation outcome. For the polystyrene beads, the experimentally measured mobility ratios closely matched theoretical predictions and showed good reproducibility. This demonstrates that the method is robust and could reduce the need for extensive trial-and-error tuning.

For biological cells, the results showed greater variability, mainly due to natural differences in cell size and properties. Nevertheless, the measured mobility ratios were within the expected range, and the method still provided useful insight into separation behaviour. Cells with very similar properties, such as K562 and Jurkat cells, were shown to be difficult to separate, which is an important result in itself.

It was further found that adding a dense medium (iodixanol) had little effect on the acoustic behaviour of the cells, while it significantly affected the reference particles. This highlights how differently synthetic particles and living cells can respond to acoustic forces.

Overall, this work shows that the mobility ratio is a practical and experimentally accessible tool for characterising acoustophoretic separations. With further development, it could help make label-free cell separation faster, more predictable, and easier to use in research and clinical applications.

• author: Wigren, Sofie
• supervisor: Thierry Baasch; Andreas Lenshof; Amalnath Manindranath
• organization: Division for Biomedical Engineering
• alternative title: Extraktion av materialegenskaper för optimiserad akustoforesbaserad partikel- och cellseparation med hjälp av flödescytometridata
• course: BMEM01 20261
• year: 2026
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: acoustophoresis, microfluidics, cell separation, flow cytometry, mobility ratio
• language: English
• additional info: 2026-03
• id: 9220879
• date added to LUP: 2026-02-10 09:56:12
• date last changed: 2026-02-10 09:56:12