We have strange things going on in our MCF-10A flask. I am wondering if the seeding density could be the cause. We seed them at 3000-4000 cells/cm2 and passage them 2 times a week. But 1). The luminal markers they are said to express (for example E-cad) are expressed only in tiny epithelial islands (tightly packed cobble stone cells) but they are surrounded by a sea of more spindle shaped cells that are not expressing these markers) they are not forming nice looking acini when placed in Matrigel. I wonder if seeding density could induce epithelial-mesenchymal transition or if the high passage number of the cells provided by ATCC could be the cause of this problem.
Regarding seeding density, a subcultivation ratio of 1:3 to 1:4 is recommended by ATCC, which basically doesn't mean a lot. It has been reported that "In response to low cell density, MCF10A cells suffer spontaneous morphologic and phenotypic EMT-like changes, including cytoskeleton reorganization, vimentin and Slug up-regulation, cadherin switching, and diffuse cytosolic relocalization of the catenins." (Sarrio et al. 2008). It was later proposed that MCF-10A have a gene expression profile similar to luminal progenitor cells but they also undergo spontaneous epithelial-mesenchymal transition (EMT). Removal of the more fibroblastic cells by selective trypsinisation (removal of the cells that float the first upon trypsinization) or isolation of EpCAM+/Cd49f+ cells by FACS restored a more purified population but this EMT happened again in this pure population (Sarrio et al. 2012).
I wonder if at high density, the MCF-10A can go back the opposite way (mesenchymal-epithelial transition)? Does increasing density upon cell growth reverse this effect (I can't see that in my flask!)? Is a high seeding density necessary to keep their luminal characteristics?
Has anyone had to deal with such a problem? Is it that everybody working on MCF-10A in 3D has a source of low passage cells?
Does anyone study the luminal side of MCF-10A in 2D or 3D? Are you witnessing some plasticity of these cells?
Hi there!
I've worked with MCF10a for almost 4 years now, and seeding density is very important. I've found that at low density MCF10a shows a more mesenchymal phenotype expressing N-cadherin and look more spindle-like. As cells grow, they become more epithelial. I believe as long as cells are in contact with each other they express their luminal phenotype. When treating with TGF-beta, cells must have roon to stretch to express N-cadherin. Cells that are too densely seeded are not able to induce N-cadherin expression after treatment.
Low passage also is important, however I think you will notice when MCF10a cells are too passaaged, as they start to look grainy and do not grow normal.
When you maintain your cells, MCF10a can be confluent for only a day or so and will still continue to grow when split. I plate MCF10a anywhere from 5k/cm^2 (equivalent to ~290k cells in a 10cm dish: results in sparse density and little to no cell contact) to 100k/cm^2 (equivalent to ~2.1M cells in a 6cm dish; results in 95% confluency).
Hope this helps and good luck in your studies.
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