Rinse out the culture medium with water, then do a slow-freeze in a normal fridge's freezing compartment, before a slow-thaw outside the freezing compartment. This slow-freeze/slow-thaw process will rupture both cytoplasmic and nuclear membranes for more effective post-processings, whether mechanically, chemically or biologically.

 Hi Ali, 

I am looking to preserve as much as possible the proteins and get rid of any cellular components. We would like to use the cells-made proteins in further experiments, therefore, using aggressive decellularization methods might affect the quality of the proteins. 

Hi Ali, 

thanks for your suggestions. We want to keep as much proteins as possible, while removing DNA as much as possible, as we want to recellularize. So aggresive methos such as SDS, high temperature, mercapthoethanol would be something to avoid.  We are following standard and published protocols, but so far out attempts did not work out. Nuclei are still present and on top of that, ECM detaches from the substrate. We have done a slow freeze thaw cycle (as described above by Kwok-Hung). We can see damaged nuclei, but still present, therefore we will do 3 times this cycle and further proceed with the decellularization protocol.  However, so much manipulation will just increase the likeliness for the ECM sheet to detach. 

We will continue with improving our protocol. But if any of you has any other suggestion, we are happy to try in on. 

Thanks again for all your valuable support. 

Silvia

We are working with human adipose derived stem cells and human smooth muscle cells. We are following the protocol described by Rakian et al. Stem Cell Research & Therapy (2015) 6:235, Native extracellular matrix preserves mesenchymal stem cell “stemness” and differentiation potential under serum-free culture conditions, the same desribed by Cukierman et al. Current Protocols in Cell Biology (2002) 10.9.1-10.9.15. It seems a very simple procedure, but in our hand we reduce  the amount of DNA by just 50%. We need more ....