Optimization of Culture Medium for the Isolation and Propagation of Human Breast Cancer Cells from Primary Tumor Biopsies

Abstract

Breast cancer cells from patients hold an important role in antigen production for immunotherapy, drug testing, and cancer stem cell studies. To date, although many studies have been conducted to develop protocols for the isolation and culture of breast cancer cells from tumor biopsies, the efficiencies of these protocols remain low. This study aimed to identify a suitable medium for the isolation and propagation of primary breast cancer cells from breast tumor biopsies. Breast tumor biopsies were obtained from hospitals after all patients had given their written in-formed consent and were cultured according to the expanding tumor method in 3 different media: DMEM/F12 (Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12) supplemented with 10% FBS (Fetal bovine serum) and 1% antibiotic-antimycotic (Medium D); Medium 171 supplemented with 1X MEGS (Mammary Epithelial Growth Supplement) and 1% antibiotic-antimycotic (Medium M); or a 1:1 mixture of Medium D and Medium M (Medium DB). The cell culture efficiency was evaluated by several criteria, including the time of cell appearance, cell morphology, capability of proliferation, cell surface marker expression, ALDH (Aldehyde dehydrogenases) activity, karyotype, and tumor formation capacity in immune-deficient mice. Notably, primary cancer cells cultured in Medium DB showed a high expression of breast cancer stem cell surface markers (including CD44⁺CD24⁻ and CD49f⁺), low expression of stromal cell surface markers (CD90), high ALDH activity, an abnormal karyotype, and high tumor formation capacity in immune- deficient mice. These findings suggested that Medium DB was suitable to support the survival and proliferation of primary breast cancer cells as well as to enrich breast cancer stem cells.