I'm trying to grow PDX-derived cells from a SCCOHT model and I need them to survive short-term (7-14days) ex vivo
After tumor digestion I platted 7,000,000 cells in a 10cm ULA dish
In the following day they were forming spheroids but the spheroids were aggregating/clumping (First picture)
I filtered through a 40uM strainer and collect the portion that remained in the filter, washed with PBS, added 0.5mL of trypsin for 40s, neutralized with 1mL of 10% FBS media and them diluted in the serum reduced media (Counted with trypan blue and had >90% viability - Second picture)
I repeated this one more time after 3 days but they keep forming these giant aggregates every 2-3 days
I'm unsure if it is worse to separate them into single cells and lose the cell-cell contact or to let them grow in aggregates of spheroids
Does anyone know how to procced in this situation?
I digested the PDX tissue in Dispase/DNAse for 30min, filtered through a 100uM strainer, lysed the RBCs, minimized the debris with Ficoll and them platted in Advanced DMEM + 5% FBS
You can add some BSA in the culture medium and reduce motions. However, practically, it is almost impossible to get perfect spheroids as spheroids tend to aggregate when cultured into a liquid. Some scientists culture them in a gel to get rid of aggregation.
To reduce (it still does not prevent) sphere aggregation, especially directly after cell isolation from tumor tissue, we usually seed a maximum of 750000 cells per 10 cm dish in 12 ml of medium (depends a bit on the cells, and how fast they proliferate. We used to use a higher cell density before, but reduced it due to sphere aggregation). If you want to have a look, here's the protocol we use. Even if it's about GBM cells from patient material, we use the same protocol for cell isolation and culture of PDX-derived cells. Article Isolation and Culture of Primary Glioblastoma Cells from Hum...
Hope that helps a bit.
Thank you all for your insights, I appreciated
I will try to seed at lower density and triturate it mechanically with a 21G needle as you suggested
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