Study on Isolation and Culture of Primary Lung Fibroblasts from Newborn Mice by Improved Tissue Adhesion Method
10.3969/j.issn.1671-7414.2024.06.036
- VernacularTitle:改良组织贴壁法分离培养新生乳小鼠原代肺成纤维细胞的研究
- Author:
Xiaodan ZHENG
1
;
Ting WANG
;
Yuhai HU
Author Information
1. 武汉市汉口医院检验科,武汉 430010
- Keywords:
lung fibroblast;
primary culture;
tissue adhesion method;
flow cytometry staining
- From:
Journal of Modern Laboratory Medicine
2024;39(6):206-210
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish an improved simple and efficient method for isolation and culture of primary lung fibroblasts(LFB)from neonatal mice,and study their growth characteristics in vitro.Methods The lungs of 3-days-old mice or 8-weeks-old mice were taken under sterile conditions,and the stromal tissue was cut into 1mm3 tissue mass.High-glucose DMEM culture medium containing 10g/dl fetal bovine serum(FBS)or 20g/dl FBS was used for tissue adhesion culture.The LFB was purified by the differential time adhesion method,and the growth morphology and adherence state of the cells were observed dynamically under an inverted microscope.The primary LFB was identified by flow cytometry.The activity of the third-generation cell after culture was detected by CCK-8 assay.Results Lung tissues in mice at 3 days of age and 20g/dl FBS concentration cultured by the improved tissue mass adherent cultured method began to grow radially to the periphery on the 2nd day.On the day 7th,the cells growth density reached 90%,and the cell morphology was as a spindle.The CD140a positive and CD45 negative cells reached more than 90%after purification by differential time adhesion method,and the cells still maintain good cell activity after cultured for 3 generations.Conclusion The improved tissue adhesion method can obtain a large number of purified mice LFB with good activity simply and efficiently,which lays a foundation for the study of lung inflammation,tumors and in vitro efficacy of drugs.
