This is in contrast to the proposed method of PP-50 mediated trehalose delivery [27]. In the current study, the techniques for the cryopreservation of cells using trehalose and PP-50 developed by Lynch et al. [27] were extended to successfully preserve nucleated human cells. The Human osteosarcoma derived cell line SAOS-2 [16] and [35]

was used as a model for nucleated, adherent human cells. Unless otherwise stated, all reagents were purchased from Sigma–Aldrich (UK). Materials for the PP-50 polymer synthesis were sourced as previously described [25]. Foetal bovine serum (FBS), l-glutamine, and penicillin/streptomycin were purchased Ipilimumab supplier from Invitrogen (UK). Dulbecco’s Phosphate-Buffered Saline (DPBS), 10 × DPBS and trypsin–EDTA were purchased from Life Technologies™ (UK). The CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) was purchased from Promega (UK). The SAOS-2 cells were purchased from the European Collection of Cell Cultures. The Annexin V-FITC Apoptosis Detection Kit was purchased from BD Biosciences (UK). The synthesis and characterisation Dabrafenib datasheet of the PP-50 polymer were as previously described by Lynch et al. [25]. SAOS-2

cells were grown in tissue culture flasks containing “growth media”: Dulbecco’s Modified Eagle’s Medium – high glucose (DMEM), supplemented with 10% (v/v) FBS, l-glutamine (2 mM), penicillin (100 IU/ml) and streptomycin (100 μg/ml). At approximately 70% confluency, the cells were subcultured with trypsin (0.05% w/v) and EDTA (0.02% w/v), and were subsequently split at a ratio of 1:6. The cells were maintained Loperamide in a humidified incubator at 37 °C with 5% CO2. The cells were used between passages 4 and 20. Calcein, which is membrane impermeable, was used as a tracer for hydrophilic species delivery into the cells. The viability of the

cells was assessed using propidium iodide (PI) staining. SAOS-2 cells were seeded into 35 mm glass bottom culture dishes (PAA, UK) at 2 × 105 cells/dish, in growth media. After 48 h of incubation in a humidified incubator at 37 °C with 5% CO2, a positive control for PI staining was prepared by fixation with paraformaldehyde solution (4% w/v, in DPBS) for 10 min, followed by washing (×3) with DPBS. For the remaining dishes, the cells were washed twice with DPBS. Afterwards, the cells were incubated for 4 h in serum-free media supplemented with 0.2 M trehalose, 2 mM calcein, and with or without PP-50 (200 μg/ml), at pH 7.05. The cells were washed twice with DPBS, and incubated with growth media containing Hoechst 33342 (2 μg/ml) and PI (2 μg/ml) for 15 min. Following three washes with DPBS, the cells were imaged using a TCS SP5 inverted laser scanning confocal microscope (Leica, Germany). SAOS-2 cells were seeded into 96-well tissue culture plastic plates (Corning, UK) at 5000 cells/well. After 24 h, the cells were washed twice with DPBS at either pH 7.4 or pH 7.05.