Culturing primary cells from the nose we are constantly dealing with a loss of cultures due to persisting primary contamination after the first 3 days period (protocol according to ller L, Brighton LE, Carson JL, Fischer Ii WA, Jaspers I. Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface. 2013(80): e50646).

To deal with the persisting contamination we chose to substitute the culture media with 1% of Pen/Strep/Neo. The contamination was cleared and cells grew normal again, indicating that the contaminant was indeed bacterial. However the contamination re-appeared once antibiotics discontinued. Long term use (more than 10 days) however had the effect that the differentiation process was disturbed (cells died off once retinoic acid was supplemented to induce the differentiation process). 

In a previous version of our protocol we had supplemented our media with Gentamycin/Amphotericin (final concentration: 7.5ug/ml and 3.75ng/ml) solution until the end of the differentiation induction process without negative effects on differentiation.

Additionally we also observed the same cell lose in cell cultures which at the time point of antibiotic treatment were either partially or fully differentiated with ONE EXCEPTION.

Does anyone has an answer to why Pen/Strep/Neo may affect the cultures in a differentiated stage or the differentiation process while the use of Gent/Amp during induction clearly had no negative effect? Could there be another reason for our observation? Thanks in advance!