Does anyone have good lab practices or experience of growing primary cells without antibiotics and anti-fungal?
Recently I ordered primary airway epithelial cells (adherent) from ATCC. They provided Anti-anti (penicillin + amphotericin B solution) along with basal media. so I prepared media with anti-anti solution and re suspend cells. so I observed cells were not attaching for 2 days and 3rd day there was contamination without change of media color. it was rod shaped bacteria with cells when i saw under microscope.
So when I complaint to ATCC, they suggested me some points. One of them was not to use anti-anti solution (antibiotics), as it can cause stress to primary cells.
Now I am planning to grow primary cells without adding antibiotics, But I am scared that there will be contamination and I dont want to lose cells (I have only single vial).
Any suggestions?
Maintain sterile cultures with careful attention to sterile techniques and proper culture conditions i.e. culture optimization.
Dear Ayesha,
If there was bacterial growth even in the presence of antibiotics, and the cells were initially free of contamination, then it may be necessary to check the filters of your cell box. Infection could have occurred due to dirty filters. To do this, you need to leave the clean medium in a cell box with the plugs open for some time, and then cultivate it in a thermostat. It is also necessary to disinfect the cell box with ultraviolet light for at least 30 minutes before planting cells. If there is no contamination during cell planting, then cells can be grown without antibiotics. Alternatively, antibiotics can be added to the cell washing media and then cultured without atnibiotics. You can also try changing antibiotics, for example using gentamicin. If cell attachment does not occur, then it is possible that you need to use plastic flasks from another company.
Best wishes,
Rustem Uzbekov
Hello Ayesha Ayesha
It is always a good practice to work in cell culture without antibiotics. For this purpose, you need to maintain a good sterile environment in cell culture. With aseptic techniques in place, it is possible to grow healthy cell cultures without the use of antibiotics.
Aseptic techniques when practiced consistently, can help to ensure the sterility of all culture media and culture vessels, thereby reducing the chance of cells being exposed to contaminants and keeping cultures healthy, viable, and pure.
There are a few measures which you could follow.
1. Sterilization of all items before starting.
2. Use sterile pipettes, pipette tips and plasticware.
3. Do not touch sterile items to non-sterilized surfaces (including gloved hands to your own skin, clothes or hair).
4. Pre-sterilize all reagents and equipment.
5. Check whether the culture hood is working properly.
6. Always UV irradiate the laminar hood for 20 minutes between cell types and when finished.
7. Do not enter a cell culture facility with a gown from outside of the facility. There should be separate gowns for cell culture use only.
8. Head cap and nitrile gloves must be worn at all times when using safety cabinets.
9. The work area should always be sterile and tidy. Do not crowd the biosafety cabinet with culture wares, reagents or small equipment.
10. You should frequently clean and de-contaminate the hood, incubators, water bath and fridge.
11. Do not crowd the culture working area. Preferably one person should be working at a time in the hood.
12. Fumigate the cell culture lab once a month during the weekend.
These above measures will help you keep away contamination.
Good Luck!
Thank you Ayyaz Khan , Rustem E Uzbekov for your guidance. and many thanks Malcolm Nobre for the detailed points to be considered during cell culture.