How about trying

less digestion time and for brief period of less serum concentration.

Dear Nidhi,

You can find necessary information for establishing a good primary culture of breast cancer cells in a parallel link running on RG.

https://www.researchgate.net/post/How_to_establish_a_breast_cancer_primary_cell_line

For a detailed protocol I am sharing a combined pdf file of relevant articles. The protocol described in (Ponti D et al, Cancer Res, 2005) article is as follows.

Isolation and in vitro expansion of progenitor cells from breast tumor specimens:

Tumor specimens were obtained from consenting patients according to the Internal Review and the Ethics Boards of the Istituto Nazionale Tumori of Milan, Italy. Sixteen breast lesions, from the histologic diagnostic assessment and sampled by pathologists, were received in the Laboratory within 30 minutes of surgery and immediately mechanically disaggregated. Occasionally, enzymatic digestion was also required and tissue fragments were incubated at 37°C for 2 hours in a 1:1 solution of collagenase/hyaluronidase (Roche Diagnostics GmbH, Mannheim, Germany and Sigma-Aldrich Corp., St. Louis, MO, respectively). After filtration through a 30 μm pore filter, single cells were plated at 1,000 cells/mL in serum-free DMEM-F12 (Cambrex BioScience, Venviers, Belgium), supplemented with 10 ng/mL basic fibroblast growth factor (bFGF), 20 ng/mL epidermal growth factor (EGF), 5 μg/mL insulin, and 0.4% bovine serum albumin (all from Sigma). Cells grown in these conditions as nonadherent spherical clusters of cells (usually named “spheres” or mammospheres”) were enzymatically dissociated every 3 days by incubation in a trypsin-EDTA solution (Cambrex) for 2 minutes at 37°C. Conversely, differentiation was induced by culturing mammosphere-derived cells for 8 days on collagen-coated dishes in DMEM-F12 supplemented with 5% fetal bovine serum (Cambrex) without growth factors.

Viable, floating single cells were collected from the supernatant of confluent MCF7 cells by centrifugation at 1,200 rpm for 5 minutes and plated at 1,000 cells/mL in the growth medium described above.

Hope that helps.

But can we use growth factor for culturing cancerous cell !

Right, our procedure uses a DMEM media + 10% inactivated FCS+ 1% L-Glutamine and 1% p/s. So I suggest you to give our procedure ago.

you can also coat your flask with collagen IV for 1 hour, which will help cells to attach.

The number of seeded cells can effect as well. So for T25 seed no more than 1.5 x10^6; whilst not more 2.5 for T75.

Hope that will help

advice:surgical specimens do primary culture is very difficult, easy method is implant it subcutaneously in immunodeficient mice. If tumorigenesis is first step OK. Suggest using breast cancer patients of pleural effusion. In start, from the patient's own effusion cultivation, and not add heterogeneous FBS, only own effusion. You can also add FBS a little to effusion in other cell culture bottles at the same time, don't always move the bottles, cells need to be quiet. Watch once a day. Two days by centrifugal removing lymphocyte and blood cells change new pleural effusion. Try to be patient. Good luck.

Above suggestions are very good. One more point: the fatty tissue must be removed by differential centrifugation before digestion with enzymes. The ratio of enzymes (your digestion buffer) to size of tissue also matters. Too much digestion can lead to floating cells. If you have fibroblasts mixed with epithelial tissue, you can keep them for 1-2 days to stabilize the epithelial spheroids. Then, mild trypsinzation will loosen all cells. If you put this mixture of cells onto new flask, fibroblasts will stick first. You may have to do this few times to separate all the fibroblasts from the epithelium. All the Best!

I can send you one of my papers about the isolation of epithelial organoids from rat mammary glands.The procedure should be the same but there are little tricks that I'm more than willing to explain to you if you are interested.

Ya sure. . It will be great. .