I need to treat healthy primary human mammary epithelial cells. When I thaw the cells, I obtain a very small number of cells that appear unhealthy. I have used Media 171 and MEGS (mammary epithelial cell growth supplement). Additionally, I find it challenging to culture them on Matrigel. If you have any protocols for organoid cell culture or alternative approaches to treat these cells, please share.
Many Thanks
Hi Hanieh,
I work with lung epithelial cells, so I can only give you more general advice.
In terms of cell viability on thawing, there are several factors to consider:
Cryopreservation and thawing method can affect viability of the cells. The general aim is to thaw and wash the cells as quickly as possible so as to minimise their exposure to the DMSO in the cryopreservation media.
I personally thaw the cells with agitation in a waterbath, then transfer the entire contents to a 15mL falcon tube as soon as it's thawed. Then I centrifuge at 300xg for 5 minutes before resuspending the cell pellet in my expansion growth media and plating. The composition of the cryopreservation media can also be important.
The condition of the cells can also be important were the cells frozen from tissue digest or adherent cell culture? If thawing vials from previous cell culture, the confluence of the cells before freezing can affect their viability, and also remember that primary cells generally cannot divide indefinitely and will not recover well from high passage numbers.
In terms of culture conditions, I use Stemcell Technologies Pneumacult media for my adherent expansion culture and specialised culture model media. I know that they also have MammoCult for mammosphere culture. It's expensive, but may be worth looking into.
Hopefully this helps getting you started!
Sam
Hi Sam, thank you so much. The cell store 6 years ago with isolating technical, unfortunately, they are not so happy. I used Human Mammary Epithelial Cell Basal Medium ,but yes definitely worth trying MammoCult.
Many thanks
Hi Hanieh,
Six years should be fine, given that they were stored properly in liquid nitrogen, and didn't accidentally thaw at any point in time!
Sam