After a week multicellular aggregates and necrotic cells (probably) appears. Have you ever seen similar picture?
Thanks
Yes, Its the way the hepatocytes culture behaves . You will see after a pair of weeks that clusters of albumin positive cells will appear. Of course you will also find Ito Cells, Kupffer , endotelial and epitelial duct cells. We have published this sort of results in long term rats and human cultures ( Barbich et al)
Thank you very much. We have definitely crosscontamination by other cell types than stellate cells. But I thought that hepatocytes apears to be more polygonal and that they are not able to maintain under nonoptimized conditions in the cell culture. So they clusters, shrinks a die? Thats why we see many empty round space i the middle of the picture?
Thank you in advance
I think your contamination with other cell types is too big - did you define the initial purity somehow? The clumps with many nuclei side by side look like long-term cultured hepatocytes - hepatocytes do not simply die, but they loose their epithelial phenotype. Those cells with the long extensions could be the hepatic stellate cells which became myofibroblasts (as they should). In general this looks like a co-culture of several liver cell types and you should try to optimize the isolation procedure of the HSC to get more pure cells in the beginning.
Hepatic stellate cells, that are placed in conditions that drive proliferation and conversion to their myofibroblast phenotype, will tend to contract and mound up with time in culture. The empty spaces are areas that have cleared from the contraction/mounding process. Your photo does suggest you have some co-cultured cells as well, but the 'holes' are as above.
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