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Updated: Jul 26, 2025
Generation of Single-Cell Suspensions from Mouse Neural Tissue
Published on: July 7, 2009
Optimization of method for achieving a single-cell suspension from mouse corneas.
Xiaodong Liu1, Shengpeng Zhang1, Yi Mao2
1Eye Institute and Affiliated Xiamen Eye Center of Xiamen University, Fujian Provincial Key Laboratory of Cornea & Ocular Surface Diseases, Xiamen, Fujian, 361002, China; School of Medicine, Xiamen University, Xiamen, Fujian, 361002, China; Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, 361002, China.
Optimizing single-cell suspensions from mouse corneas is crucial for single-cell RNA sequencing (scRNA-seq). Method 2, using collagenase A and trypsin, yielded the highest cell viability and single-cell percentage for scRNA-seq.
Area of Science:
- Ophthalmology
- Molecular Biology
- Biotechnology
Background:
- Single-cell RNA sequencing (scRNA-seq) is vital for understanding tissue biology in health and disease.
- Obtaining high-quality single-cell suspensions is a significant challenge in scRNA-seq protocols.
- Chronic eye diseases require detailed cellular analysis, often utilizing scRNA-seq.
Purpose of the Study:
- To optimize a method for digesting whole mouse corneas to obtain single-cell suspensions.
- To evaluate five different digestion protocols for their efficacy in preparing corneal single-cell suspensions for scRNA-seq.
Main Methods:
- Five distinct protocols involving enzymatic digestion (trypsin, collagenase A, TrypLE) and mechanical disruption were tested.
- Cell viability, single-cell percentage, cell yield, and epithelial cell rates were assessed using Trypan blue staining and immunofluorescence.
- RNA integrity was measured using the Agilent 2100 bioanalyzer.
Main Results:
- Method 2 (5 mg/ml collagenase A followed by trypsin) demonstrated superior performance with over 90% cell viability, 89.53% single-cell percentage, and a high cell yield (1.05 × 10^5).
- Other methods exhibited lower cell viability, higher cell agglomeration rates (up to 20%), and reduced epithelial cell percentages.
- Method 1 yielded the lowest cell quantity and viability, rendering it unsuitable for scRNA-seq.
Conclusions:
- The optimized protocol using 5 mg/ml collagenase A and trypsin (Method 2) is highly effective for preparing mouse corneal single-cell suspensions.
- This method meets the stringent requirements for successful downstream single-cell RNA sequencing applications.
- The findings provide a reliable protocol for researchers studying ocular tissues at the single-cell level.
