人类小鼠原代肺成纤维细胞分离方法

Primary human/mouse lung fibroblast isolation

人类/小鼠原代肺成纤维细胞分离方法

参考文献(H: 1.Katharina Heinzelmann et al. Am J Physiol Lung Cell Mol Physiol. 2018; 2. Claudia A Staab-Weijnitz et al. Am J Respir Crit Care Med. 2015. FK506-Binding Protein 10, a Potential Novel Drug Target for Idiopathic Pulmonary Fibrosis; M: Isolation and characterization of mouse fibroblasts, Benjamin L. Edelman and Elizabeth F. Redente)

Materials:

材料：

1. Digestion solution: a).Liberase TM (5401119001, Merck) solution,

2.5mg/ml (HBSS);

b).DNAse (D4263-5VL, Sigma), 2000U/ml(PBS)

消化液：a) Liberase TM 溶液（5401119001, Merck）：溶于HBSS（终浓度2.5mg/ml）

b) DNAse溶液（D4263-5VL, Sigma）：溶于PBS（终浓度2000U/ml）

2. Nylon filters 40/70 µm

3. Complete medium (Mouse: DMEM/F12 + 20% FBS + 1% Penicillin/Streptomycin + 0.1% Amphotericin B + 1% Glu; Human: DMEM/F12 + 20% FBS + 1%

Penicillin/Streptomycin + 0.1% Amphotericin B)

4. Knife (disposable)

5. Syringe

6. PBS

Digestion solution:

Steps: (Mouse from 1-13, Human from 7-13)

步骤：

1. Weight the mouse, calculate the amount of anesthesia, Xylazin : Kelamin : NaCl =

1:4:5, 100ul/10g weight;

称重小鼠后麻醉

2. Inject and wait 10min till the mouse sleep

腹腔注射后等待10分钟

3. Open abdomen and cut the artery of the kidney

打开腹腔切断肾动脉

4. Use syringe insert the heart and rinse with 15ml PBS until it’s white

注射器灌注15ml PBS后插入心脏冲洗至肺部变白

5. Take out the lung and heart in the tube with ice

将心脏和肺取出置于冰盒中保存

6. Wash with 80% ethanol in 10s

在80%酒精中漂洗10秒

7. Wash with PBS 3 times, 10min (Mouse); PBS 3 times, 30min (Human)

PBS洗3次，每次10分钟（老鼠），每次30分钟（人），清洗时保存于冰盒中

8. Dissected into pieces of 1cm2 in size and digested by digestion solution at 37°C for 1

hours (shake vigorously every 15min)

使用一次性无菌刀片将组织切成小块，并加入消化液在37度水浴锅中静置1小时（每15分钟猛烈上下摇晃试管）

9. Samples were filtered through nylon filters with a pore size of 70 µm

使用70微米过滤器过滤标本

10. Centrifuged at 400 g, 4°C for 5 minutes, resuspended in complete medium

400 g, 4°C, 离心5分钟，离心后加入完全培养基重悬

11. Samples were filtered through nylon filters with a pore size of 40 µm

使用40微米过滤器过滤标本

12. Centrifuged at 400 g, 4°C for 5 minutes, resuspended in complete medium

400 g, 4°C, 离心5分钟，离心后加入完全培养基重悬

13. Medium was changed after 2 days (first check) and cells were split after reaching a

confluence of 80–90%. For the present study, phLF were used in passages 4-9.

2天后检查细胞状态并换液，当密度达到80–90%时可传代，原代成纤维细胞最佳使用为4-9代。