I purchased two CRISPR K/O pools (breast cancer cell lines MCF7’s and ZR-75-1s) from a company of my gene of interest. I sorted using flow assisted cell sorting at one cell per well in a 96 well plate (as the cells have no selectable marker for the K/O).

I used DMEM high glucose, 15% FBS, 1% penicillin/streptomycin, 1% L-glutamine and 10% conditioned media from the pooled population. For MCF7s cells this protocol worked really well and I developed many clonal populations over approx 4 weeks.

But my problem is with the ZR-75-1s. Although this is naturally a slower growing cell line than MCF7, I have seen little/ very strange growth from the sorted single cells (please see photo example attached). It has now been 6 weeks since the cells were sorted into the 96 wells.

After the first five weeks seeing no growth in the sorted cells, I have altered the media from DMEM high glucose, 15% FBS, 1% penicillin/streptomycin, 1% L-glutamine, 1nM estradiol and 10% conditioned media from the pooled population to 20% FBS and adding 0.1mM MEM non-essential amino acids and 1nM sodium pyruvate to try and encourage growth.

Over the past week I have seen no noticeable growth in any of the surviving cells…

Would anyone have any suggestions as to what I could do to help the cells start to grow ?

Has anyone also tried clonal expansion with ZR-75-1s ?

Would it be a good idea to also supplement with insulin ?

I know ZR-75-1 cells can also be cultured in RPMI, could this be causing my issue (although I have grown populations of ZR-75-1s in DMEM before) ?

Can anyone explain the strange cell phenotype or what could be causing this ?

Any suggestions would be very much appreciated !

Thank you for your time :) !