Eye 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.
The Affiliated Hospital of Guizhou Medical University, Guiyang, 350000, Guizhou, China; Eye 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.
Exp Eye Res. 2023 Aug;233:109544. doi: 10.1016/j.exer.2023.109544. Epub 2023 Jun 17.
The single-cell RNA-sequencing (scRNA-seq) technique is used to explore the biological characteristics of tissues under pathological and physiological conditions that include certain chronic eye diseases. Harvesting of single-cell suspensions is one challenge inherent to scRNA-seq procedures. This study aimed to use an optimized method to digest a whole mouse cornea to harvest single-cell suspensions. We utilized five different mouse cornea digestion methods to obtain single-cell suspensions: (1) 5 dissected mouse corneas were cut into pieces (∼0.5 mm) and digested in trypsin for 10 min, and this digestion was repeated for 10 cycles; (2) 5 dissected mouse corneas were cut into pieces and incubated with 5 mg/ml collagenase A at 37 °C for 1h and then further digested in trypsin at 37 °C for 10 min; (3) used the same approach as that used in method 2, but the second digestion step was performed in TrypLE for 20 min; (4) used the same approach as that used in method 2, but the concentration of collagenase A was 2 mg/ml and the incubation time was 2h; (5) used the same approach as that used in method 3, but the corneas were incubated in 2 mg/ml collagenase A at 37 °C for 2h. Trypan blue staining was used to calculate the cell viability and agglomeration rate. The cell types and percentages were determined using immunofluorescence staining. RNA integrity number (RIN) was measured by Agilent 2100. Method 1 showed the lowest cell yield (0.375 × 10), epithelial cell percentage, and less than 70% cell viability, thus not a proper protocol. Method 2 showed the highest cell viability (over 90%), percentage of single-cell (89.53%), and high cell quantity (1.05 × 10). Method 3 had a significantly lower cell viability (55.30%). Cell agglomeration rates of method 4 and 5 reached up to 20% and 13%, and with lower cell viability (72.51%, 59.87%, respectively) and decreased epithelial cell rate compared to method 2 and 3. The results suggest that method 2 (5 mg/ml collagenase A and trypsin) is a preferred protocol for digesting mouse cornea to obtain single-cell suspension which achieves the criterion of single-cell RNA sequencing.
单细胞 RNA 测序 (scRNA-seq) 技术用于探索包括某些慢性眼病在内的病理和生理条件下组织的生物学特性。单细胞悬液的采集是 scRNA-seq 程序固有的挑战之一。本研究旨在使用优化的方法消化整个小鼠角膜以收获单细胞悬液。我们利用五种不同的小鼠角膜消化方法获得单细胞悬液：(1) 将 5 个分离的小鼠角膜切成小块（约 0.5mm），并在胰蛋白酶中消化 10 分钟，重复该消化 10 个周期；(2) 将 5 个分离的小鼠角膜切成小块，在 37°C 下用 5mg/ml 胶原酶 A 孵育 1 小时，然后在 37°C 下进一步用胰蛋白酶消化 10 分钟；(3) 采用与方法 2 相同的方法，但第二步消化在 TrypLE 中进行 20 分钟；(4) 采用与方法 2 相同的方法，但胶原酶 A 的浓度为 2mg/ml，孵育时间为 2 小时；(5) 采用与方法 3 相同的方法，但将角膜在 37°C 下用 2mg/ml 胶原酶 A 孵育 2 小时。台盼蓝染色用于计算细胞活力和聚集率。免疫荧光染色用于确定细胞类型和百分比。RNA 完整性数 (RIN) 通过 Agilent 2100 测量。方法 1 显示出最低的细胞产量（0.375×10）、上皮细胞百分比和低于 70%的细胞活力，因此不是合适的方案。方法 2 显示出最高的细胞活力（超过 90%）、单细胞百分比（89.53%）和高细胞数量（1.05×10）。方法 3 的细胞活力显著降低（55.30%）。方法 4 和 5 的细胞聚集率分别高达 20%和 13%，且细胞活力（分别为 72.51%、59.87%）和上皮细胞率低于方法 2 和 3。结果表明，方法 2（5mg/ml 胶原酶 A 和胰蛋白酶）是一种优选的方法，用于消化小鼠角膜以获得单细胞悬液，该方法达到单细胞 RNA 测序的标准。
