Optimization of a Clinically Relevant Chemical-Mechanical Tissue Dissociation Workflow for Single-Cell Analysis

Area of Science:

• Biotechnology
• Cell Biology
• Biomedical Engineering

Background:

• Obtaining single-cell suspensions from complex tissues is a significant challenge in single-cell analysis.
• Current methods are time-consuming and involve multiple steps.
• This study addresses the need for rapid, clinically relevant tissue preparation.

Purpose of the Study:

• To develop and optimize a rapid chemical and mechanical protocol for single-cell suspension preparation.
• To evaluate the efficiency and viability of the developed protocol for complex tissues.
• To establish a foundation for automated single-cell diagnostic workflows.

Main Methods:

• Investigated various chemical reagents (collagenase, pronase, hyaluronidase) in HBSS solution.
• Utilized confocal microscopy, hemocytometry, and quantitative flow cytometry for dissociation analysis.
• Incorporated controlled mechanical force alongside chemical treatment.
• Performed physical modeling simulations using COMSOL Multiphysics.

Main Results:

• A 15-minute protocol was developed for dissociating frozen bovine liver biopsy cores.
• Optimal dissociation achieved using 1% type-1 collagenase with pronase or hyaluronidase.
• Achieved 37-42% dissociation efficiency with >90% cell viability.
• Combined chemical and mechanical methods dissociated 93 ± 8% of the tissue into single cells.

Conclusions:

• Effective tissue dissociation requires a combination of chemical and mechanical approaches.
• The developed protocol offers a rapid and efficient method for preparing single-cell suspensions.
• This research supports the development of automated workflows for single-cell diagnostics.