In this study, we characterize prime Ren human cells of breast epithelial outgrowth of a tumor tissue directly without protease digestion. PI-103 This prim Ren cultures HBCEC k Nnte serve as a specific approach to the treatment of patients with cancer individuallydesigned optimization. Zus Tzlich cultures obtained HBCEC repr Sentieren representative features of tumor cells, as opposed to limited cell division are held by normal HMEC, providing a platform for investigating potential testing of new therapeutic strategies. Derived materials and methods each breast tumor cell cultures from different tissues of small pieces of 8 patients with breast cancer were w Collected during surgery and pathology are characterized by ductal carcinomas. Written informed consent was obtained from each patient for the use of the individual biopsy material, and the study was the 15th of the Institutional Review Board, Project # 3916 Approved in June 2005.
Tissue samples were placed in PXD101 small Bl Cke about 1 mm3 cut and washed extensively in PBS to remove blood cells and cell debris. After a test negative for HIV-1, hepatitis B and C, bacteria, yeasts and fungi are each the tissues of breast tumors with plastic dishes uncoated raw serum-free cell were incubated mammary epithelial growth with 52 g / ml bovine extract pituary, 0 erg Complements. 5 g / ml hydrocortisone, 10 ng / ml recombinant human epidermal growth factor and 5 g / ml of recombinant human insulin in a humid atmosphere at re 37th Replaces H half The cell culture medium was every four days, and the other is H Half was used as a conditioned medium. Under these conditions tumorderived an outgrowth of prime Ren cells was observed, which are adh Rent tumor tissue Bl Cke and among themselves.
Subconfluent in the growth phase of pieces of tumor tissue were removed from the culture and in a box Te separate culture further outgrowth prim Re tumor cells erm Equalized. Other tumor cells were derived using appropriate dosages. Normal human mammary epithelial cell cultures of primary Rkulturen normal human mammary epithelial cells were isolated from a Caucasian female age 50 and supplied commercially from BioWhittaker Inc. passage 7 of culture. HMEC were tested positive cytokeratins 14 and 18 and negative for cytokeratin 19, respectively. They have been tested and performance tested negative for HIV-1, hepatitis B and C, mycoplasma, bacteria, yeast and fungi.
HMEC were sown at 4500 cells/cm2 T and was raised in the appropriate media MEBM every culture every two to three days. Under subconfluent cells were subcultured by incubation with 0. 025% / 0 Detached 01% trypsin / EDTA to the cells about 6 min/37 st. Subsequently End was the immediate addition of neutralizing L Tion is required of soybean trypsin inhibitor to the trypsin by centrifugation sp Inactivate ter. The sedimented cells were resuspended in fresh medium at approximately 4500 cells/cm2 and further into the n Cultured next issue passage.