Tissue Dissociation and Label Free Sorting Semi-Automated Platform for Single Cell Recovery

Introduction: Single cells derived from tissue dissociation of primary tissues and cell aggregates are crucial for numerous downstream single-cell analysis studies and tissue engineering applications. To address these challenges, our group previously established a protocol utilizing microfluidic devices to efficiently digest primary tissue into single cells through enzymatic and mechanical digestion, reducing tissue dissociation processing time by 50% compared to standardized protocols. Yet, in our current system, liberated single cells from the tissue may undergo unnecessary damage and destruction due to recirculation in the enzyme. To mitigate this risk, we developed an integrable acoustophoretic device that diverts cells from the digestion device into a recovery stream via acoustic radiation forces for collection, thereby minimizing recirculation damage and improving cell yield. While acoustophoretic devices have traditionally been standalone tools for particle and cell separation, few studies have explored their potential for automation. In this research, we present a semi-automated system for label-free sorting of single cells from dissociated tissue to further increase our total single-cell yield compared to traditional tissue digestion methods and advance the development of acoustophoretic automation in biomedical research.

Materials and

Methods: A peristaltic pump served as the fluid delivery system for both the tissue dissociation and acoustophoresis microfluidic devices. Given that mixing within the acoustofluidic device takes place under peristaltic flow conditions, we quantified the extent of mixing within the acoustic device by measuring the concentration of mixed dye fluid between the recovery and sample outlets using spectrophotometry and determined that the optimal flow rate for minimal mixing was 10 mL/min for both sample and recovery stream inlets. Once we established the optimal flow rates, we conducted experiments using the MCF-7 human breast cancer cell line and 20-μm diameter polystyrene beads (Sigma Aldrich) to evaluate the separation efficiency of the acoustofluidic device with the acoustic field switched on versus off. Additionally, murine liver tissue from 5-month-old wild type mice was dissociated in the integrated platform at an optimal flow rate of 10 mL/min. We compared the automated dissociation-sorting method with control digestion techniques commonly employed in standard tissue dissociation and our lab’s microfluidic dissociation protocols. To control the speed and orientation of the enzyme within the system and to activate the acoustic field at specific intervals, we employed custom-written MATLAB code. Aggregates and single cells dissociated from the murine liver tissue were sorted using the integrated acoustophoresis device and then further dissociated and filtered using our lab’s integrated dissociation and filtration (IDF) device to enhance single cell yield for cell identification via Flow Cytometry (Novocyte 3000). Flow cytometry was also used to determine single cell yield recovery for all tested conditions.

Results, Conclusions, and Discussions: The absorbance study revealed similar mixing percentages of the liquid dye (22.7%) between the recovery and sample streams of the acoustophoresis device, compared to non-acoustic mixing of MCF-7 cells (21.1%) and 20μm polystyrene beads (18.7%). When the acoustic field was activated during a single pass test, the acoustofluidic device's separation efficiency yielded approximately 2x and 3x fold recovery of MCF-7 cells and polystyrene beads, respectively, compared to when the acoustic field was off. After determining the separation efficiency of the acoustic field for a single pass, we investigated the device's ability to recover particles from a sample for various acoustic time intervals. As expected, longer exposure times to the acoustic field (10, 20, or 30 seconds) resulted in a higher percentage of particle recovery compared to when no acoustic field was present. To further validate the acoustic results obtained with MCF-7 single cells and 20μm polystyrene beads, murine liver tissue was utilized in the final version of the semi-automated platform. The tissue to single-cell acoustic sorting platform yielded a higher number of total single cells compared to when the acoustic field was off, control digestion conditions, and current manual collection protocols used in our lab. Additionally, the recovery of hepatocytes, white blood cells, and endothelial cells was higher with the acoustic field compared to all previously mentioned methods. The results of the single cell, polystyrene beads, and liver tissue dissociation experiments provided further confirmation that integrating the polymer acoustophoresis device with our lab's tissue dissociation device enables the sorting of label-free single cells based on their size, density, and compressibility. Ultimately, these findings exemplify a practical application of a hands-free tissue to single cell acoustophoresis system for single-cell and particle recovery.

Acknowledgements (Optional): I would like to thank the Transgenic Mouse Facility (TMF) at UC Irvine and there workers for providing us with fresh mice for the experiments.