Volume 16, Issue 2, Pages 181-190 (February 2014) Human mesenchymal stromal cells can uptake and release ciprofloxacin, acquiring in vitro anti-bacterial activity  Francesca Sisto, Arianna Bonomi, Loredana Cavicchini, Valentina Coccè, Maria Maddalena Scaltrito, Gianpietro Bondiolotti, Giulio Alessandri, Eugenio Parati, Augusto Pessina  Cytotherapy  Volume 16, Issue 2, Pages 181-190 (February 2014) DOI: 10.1016/j.jcyt.2013.11.009 Copyright © 2014 International Society for Cellular Therapy Terms and Conditions

Figure 1 Differentiation potential of human BM-MSCs. BM-MSCs were studied for their ability to differentiate in vitro into adipocytes (B), osteoblasts (C) and chondroblasts (D) under specific stimulation. (A) Negative control (cells grown in absence of differentiation stimuli). Cells in (A) and (B) were counterstained by 0.1% crystal violet. Cytotherapy 2014 16, 181-190DOI: (10.1016/j.jcyt.2013.11.009) Copyright © 2014 International Society for Cellular Therapy Terms and Conditions

Figure 2 Human BM-MSC sensitivity to CPX. (A) Effect of increasing concentrations (0–500 mg/L) of CPX on a 7-d cell proliferation in an MTT assay. The effect is expressed as percentage of the optical density (OD) measured in cultures that did not receive CPX (100% proliferation). Cytotoxic activity (B) was evaluated as cell viability at 24 h of treatment with CPX up to the dose of 2500 mg/L. Box shows IC50 and IC90 values as mean ± standard deviation of three independent experiments. Cytotherapy 2014 16, 181-190DOI: (10.1016/j.jcyt.2013.11.009) Copyright © 2014 International Society for Cellular Therapy Terms and Conditions

Figure 3 HPLC analysis of BM-MSCsCPX-CM and BM-MSCsCPX-LYS. The release of CPX in BM-MSCsCPX-CM and the amount of CPX in the BM-MSCsCPX-LYS were analyzed by means of HPLC. Chromatograms recorded from analysis of the BM-MSCsCPX-CM (E) and BM-MSCsCPX-LYS (D) show an elution profile with a peak comparable to that of pure CPX at 100 mg/L (B). (A) Profile of BM-MSCs-CM cultured in the absence of CPX. The lysate of untreated cells BM-MSCs-LYS did not show the presence of CPX (C). Cytotherapy 2014 16, 181-190DOI: (10.1016/j.jcyt.2013.11.009) Copyright © 2014 International Society for Cellular Therapy Terms and Conditions

Figure 4 Bioassay of BM-MSCs-CM and BM-MSCsCPX-LYS performed on E. coli, S. aureus and P. aeruginosa. BM-MSCsCPX-CM was evaluated on E. coli by agar well diffusion assay (A). Plate 1: CPX standard (2, 1 and 0.5 mg/L); plate 2: CPX standard (0.25, 0.125, 0.06 and 0.03 mg/L); plate 3: control BM-MSCs-CM (white arrow) and conditioned medium obtained from BM-MSCs after CPX priming (black arrow). Antibacterial activity of BM-MSCsCPX-LYS was evaluated on E. coli (B), S. aureus (C) and P. aeruginosa (D) by microdilution method. Panel B: row B = serial 1:2 dilution of BM-MSCs-LYS; row C = serial 1:2 dilution of BM-MSCsCPX-LYS; rows D and E, duplicate serial 1:2 dilution of standard CPX (starting stock solution 1.0 mg/L; MIC at 1:64 dilution = 0.0156 mg/L) performed in DMEM–low glucose with 10% FBS (row D) and in cation-adjusted MH broth (row E); Panel C: row B = serial 1:2 dilution of standard CPX (starting stock solution 0.5 mg/L; MIC at 1:2 dilution = 0.25 mg/L); row C = serial 1:2 dilution of BM-MSCs-LYS; row F = serial 1:2 dilution of BM-MSCsCPX-LYS; Panel D: row B = serial 1:2 dilution of standard CPX (starting stock solution 0.5 mg/L; MIC at 1:2 dilution = 0.25 mg/L); row C = serial 1:2 dilution of BM-MSCs-LYS; row F = serial 1:2 dilution of BM-MSCsCPX-LYS. Control = bacterial growth in medium without CPX. Cytotherapy 2014 16, 181-190DOI: (10.1016/j.jcyt.2013.11.009) Copyright © 2014 International Society for Cellular Therapy Terms and Conditions

Figure 5 Direct anti-microbial activity of BM-MSCsCPX on E. coli growth. The inhibition of growth of E. coli was evaluated by co-culturing bacteria with increasing numbers of BM-MSCsCPX. Results are expressed as percentage of viable bacterial counted in co-cultures with 50,000 cell not primed with CPX (100% vitality corresponding to approximately 3 × 109 CFU/mL). A number of 6250 BM-MSCsCPX produced a complete visible inhibition of bacterial growth mimicking an MIC value. Cytotherapy 2014 16, 181-190DOI: (10.1016/j.jcyt.2013.11.009) Copyright © 2014 International Society for Cellular Therapy Terms and Conditions