An Modified Primary Culture Method of Breast Cancer-associated Fibroblasts

Abstract

Objective To establish a rapid and high efficient method of primary culture of BCAFs by improving primary culture methods of common breast cancer-associated fibroblasts (BCAFs). Methods We took cancerous tissue samples from the patients diagnosed as invasive breast cancer. All samples were randomly divided into two groups: the modified group and the conventional group with same size and quality. The modified group was digested with a modified enzyme digestion method (typeⅡcollagenase+hyaluronidase digestion for 10 min). The conventional group was digested with conventional enzymatic digestion method (typeⅠcollagenase digestion for 8h). The total number of cells obtained, the activity of adherent cells after 48h culture and the purity of BCAFs between two groups were compared by cell counting, MTT and immunofluorescence. The characteristics of primary BCAFs were observed under inverted microscope. Results The amount of cells, the activity of adherent cells after 48h culture and cells purity obtained by the modified enzyme digestion method were all higher than those by the conventional enzyme digestion method (P < 0.001). The primary BCAFs obtained were spindle-shaped or spindle-shaped, with many cytoplasmic particles and irregular cell arrangement. Conclusion A rapid and high efficient primary culture method of BCAFs is established successfully, as the foundation for the later study of its function in breast cancer.

Keywords

• breast neoplasm
• fibroblasts
• cell culture techniques
• primary culture