Thein vitroeffects of antibiotics on cell viability and gene expression of equine bone marrow‐derived mesenchymal stromal cells

Abstract

To investigate the effects of commonly used antibiotics on cell viability and gene expression of equine bone marrow-derived mesenchymal stromal cells (MSC) in vitro. Bone marrow-derived MSC were cultured in media containing gentamicin, amikacin, penicillin, enrofloxacin or ceftiofur at concentrations of 50, 100, 200 and 500 µg/ml. The alamarBlue fluorescence assay was used to assess cell viability over 48 h. After 5 days the cells were released and lysed prior to RNA extraction and reverse transcription. RNA levels were assessed using spectrophotometry and quantitative PCR was used to analyse gene expression of COL1A2, COL5A1, TNC, TNFα, CASP3, BCl2 and TGFβR2 relative to the reference gene GAPDH. Enrofloxacin produced a significant concentration-dependent reduction in cell viability at 200 µg/ml and higher concentrations (P = 0.009). Amikacin significantly reduced cell viability at 500 µg/ml (P = 0.002). Penicillin had no effect on cell viability at the concentrations tested (P = 0.18). Gentamicin and ceftiofur showed some interaction with the assay but had no overall effect on cell viability. At 500 µg/ml gentamicin (P<0.001), amikacin (P = 0.03), enrofloxacin (P<0.001) and ceftiofur (P<0.001) caused significant reductions in RNA levels. At 50 µg/ml gentamicin (P<0.001) and amikacin (P = 0.019) reduced BCl2 expression. Enrofloxacin produced a significant increase in COL1A2 expression (P<0.001). Enrofloxacin reduced MSC viability in vitro and may require cautious use in clinical situations. Penicillin has minimal detrimental effects on MSC in vitro and its use in conjunction with MSC at implantation appears safe. Further work is needed to fully investigate the effects of gentamicin, amikacin and ceftiofur. Clinicians using local antibiotic administration should consider the potential for local toxicity as well as the need for effective concentrations of the antibiotic.