Dmem f12 glutamax 1

Mice bearing TSGs were sacrificed and TSGs were dissected free of surrounding mouse kidney. TSGs were placed in the Krumdieck tissue slicer and cut at 300-μm. Sequential sections were collected and alternating sections were frozen for histological analysis. Only tissue sections adjacent to frozen sections free of necrosis or cystic structures were used to generate single cells. Tissue slices were minced with scissors and then digested in DMEM/F12 + GlutaMAX-I™ (Invitrogen, Carlsbad, CA) supplemented with 10 % FBS and 200 U/ml Collagenase type I (Sigma-Aldrich), 1 U/ml DNase I (Invitrogen), 2 μM Y27632 (Sigma-Aldrich), and antibiotics for a total of 30 min at 37° C. The mixture was pipetted up and down every 10 min. The digested tissue was passed through a 40-μm cell filter (BD Biosciences, Bedford, MA) and the cells that passed through the filter were collected. At this point, cells were either immediately inoculated intratibially into mice or cryopreserved in DMEM/F12 + GlutaMAX-I™ supplemented with 10 % FBS and 5 μg/ml holo-transferrin (Sigma-Aldrich), 10 % DMSO and antibiotics. Prior to inoculation into mice, the cells were suspended at a concentration of 5 × 10⁵ live cells/20 μl of HBS and kept on ice until inoculation.

Adherent BM-MSCs were grown in D-MEM/F12/Glutamax-I™ (Gibco Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) in T-75 flasks until 80% confluent. Cells were then trypsinized (0.25% trypsin, Gibco Invitrogen, Carlsbad, CA) for 3 min, followed by trypsin neutralization using media containing 10% FBS. Cells were quantified using a hemacytometer, centrifuged at 180xg for 5 min at 4°C, and resuspended in sterile phosphate buffered saline (PBS) at a concentration of 3x10⁵ cells per 50 μl. This preparation of cells was then loaded into 0.3 ml low-dose U-100 insulin syringes with 29 gauge needles (Becton Dickinson, Franklin Lakes, NJ) immediately prior to IP injection.

SH-SY5Y neuroblastoma cells were grown in D-MEM/F12+GlutaMAX™-I (Dulbecco's Modified Eagle medium; GIBCO Invitrogen Corporation) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin solution (P/S; 100 U/mL) (Gibco). Cells were transfected using Lipofectamine™ 2000 (Invitrogen™, Life technologies Paisley, UK) with 4 μg of AChE-T or AChE-R cDNAs under the cytomegalovirus (CMV) promoter-enhancer (a generous gift from Prof Hermona Soreq, Institute of Life Science, Hebrew University, Jerusalem, Israel). The PCI “empty” vector (Promega, Madison, USA) served as negative control. The cells were collected for analysis 48 hours after the transfection.

Cell-free samples (n = 3) were prepared by injecting the different polymer solutions (Figure 1) into custom-made cylindrical Teflon molds (diameter: 6 mm; height 2 mm). Next, samples were UV cross-linked by exposure to 365 nm UV light (2.6 mW/cm², UVP CL-1000) for 15 minutes. After removing the hydrogels from the molds, they were incubated in DMEM/F-12+GlutaMax-1 (Dulbecco’s Modified Eagle Medium, 31331, Invitrogen, Carlsbad, California, USA) supplemented with 5% heat-inactivated fetal bovine serum (FBS, Biowhittaker, Breda, the Netherlands) and pen/strep (final concentration 100 units/ml penicillin and 100 μg/ml streptomycin, Gibco) for 24 hours at 37°C. A stress/strain curve was obtained for each hydrogel construct under unconfined compression using a Dynamic Mechanical Analyzer (DMA, Q800 TA-Instrument) to determine the Young’s modulus. The hydrogel constructs (three for each condition) were subjected to a preload force of 0.001 N and subsequently compressed with a force ramp rate of 0.5 N/min and an upper force limit of 1.5 N. The Young’s modulus was calculated as the initial slope (around 2% strain) of the stress/strain curve.