Conclusions
Understanding cellular dissociation in ex vivo tissues is essential to the development of clinically relevant dissociation workflows. Controlled mechanical force in combination with chemical treatment produces high quality tissue dissociation. This research is relevant to the understanding and assessment of tissue dissociation and the establishment of an automated preparatory workflow for single cell diagnostics.
Methods
Frozen bovine liver biopsy cores were normalized by weight, dimension, and calculated cellular composition. Various chemical reagents were tested for their capability to dissociate the tissue via confocal microscopy, hemocytometry and quantitative flow cytometry. Images were processed using ImageJ. Quantitative flow cytometry with gating analysis was also used for the analysis of dissociation. Physical modeling simulations were conducted in COMSOL Multiphysics.
Results
A rapid method for tissue dissociation was developed for single-cell analysis techniques. The results of this study show that a combination of 1% type-1 collagenase and pronase or hyaluronidase in 100 U/µL HBSS solution is the most effective at dissociating 2.5 mm thawed bovine liver biopsy cores in 15 min, with dissociation efficiency of 37-42% and viability >90% as verified using live MDA-MB-231 cancer cells. Cellular dissociation is significantly improved by adding a controlled mechanical force during the chemical process, to dissociate 93 ± 8% of the entire tissue into single cells. Conclusions: Understanding cellular dissociation in ex vivo tissues is essential to the development of clinically relevant dissociation workflows. Controlled mechanical force in combination with chemical treatment produces high quality tissue dissociation. This research is relevant to the understanding and assessment of tissue dissociation and the establishment of an automated preparatory workflow for single cell diagnostics.
Supplementary Information
The online version of this article (10.1007/s12195-021-00667-y) contains supplementary material, which is available to authorized users.