We are attempting to establish primary cell lines from patient tumor samples of head and neck squamous cell carcinoma (HNSCC). Following transport to LUMS within 30-45 minutes of surgery, the sample is minced on ice into 2-3mm pieces. The minced tissue is then incubated in disaggregation media containing 0.4mg/ml collagenase V and 0.2mg/ml Dispase II.
Our initial trial with a renal tumor sample using an overnight incubation in disaggregation media yielded a successful development of the primary cell line. However, replicating the same protocol with HNSCC samples overnight did not yield positive results. Subsequent attempts involving reduced incubation times of 30 minutes, 15 minutes, and even 5 minutes in diluted disaggregation media (with enzyme concentrations halved) did not produce the desired outcome. When we observe the cell under microscope while plating the cells, we always notice the presence of individual cells; however, these cells failed to attach to the surface and remained in a rounded shape.
Any recommendation to optimize our protocol and improve the attachment and growth of cells would be greatly appreciated.
I am also attaching the protocol we are using
- Badges
- Science topic
- Similar topics
- Microbiology
- Assays