Hi, I would appreciate professional and technical insights regarding primary cell culturing. I've been working on optimizing my primary cell culture, and while it is successful at times, there are instances where it doesn't work as expected. Since primary cultures are prone to bacterial infection, I have already used 1% P/S and cultured the cells for 48 hours. I conducted a time point assay at T=0, 4, 8, 12, 24, and 48, followed by Hematoxylin and Eosin staining. Starting from T=0, I observed the formation of debris in the culture, but for up to 24 hours, the different structures were still distinguishable. However, after 48 hours, only debris remained. Therefore, I would like to ask if anyone has any knowledge or suggestions on how to minimize debris formation and reduce bacterial infection. It is important to note that the primary culture is derived from fish obtained from a fish market and not bred in a laboratory. Thank you.
Hi. Primary culture is one of the riskiest cell culture methods. You may want to increase the antibiotics concentration to 2% during the culture, but just know that pen/strep only acts as a prophylaxis to prevent contamination. If the contamination is already present, chances are it will still grow. For this, you should try to disinfect your samples as much as possible. It would be best if you are performing the sample isolation in a BSC/laminar flow. Then straight after isolation, wash your samples with sterile water/PBS to get rid of other contaminants, and soak the sample in 70% ethanol for at least 5 minutes to disinfect the surface of the sample. And cut out the outer area as much as you could to remove any potential contaminated area. Another consideration is to use antibiotic-antimycotic instead of pen/strep, since pen/strep can’t prevent fungal contamination. Primocin is so far the best for primary culture in my opinion. You can also add the antimicrobial solutions in your washing solutions as well. Finally, do expect for primary culture to take a while for cells to be seen. Cells would normally show up after 4 days (depends on cell type).
You can try to look at this paper where they’ve isolated fibroblasts from an elephant’s ear and see how they manage culturing cells from a wildlife: https://doi.org/10.7717%2Fpeerj.4302
Hi Ahmad. Thank you for recommending the use of antibiotic-antimycotic instead of solely relying on P/S and for providing the link for further reading. I appreciate your valuable suggestion to incorporate antibiotic-antimycotic, which can help enhance the sterility and overall health of the cell culture. The additional information you shared in the provided link will be beneficial for expanding my knowledge on this topic. Thanks
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology
- Pharmaceuticals
- Drugs