Vivaspin 20 ultrafiltration unit

PMOXA₁₅-PDMS₆₈-PMOXA₁₅ was solubilized in ethanol (99.8 %) to obtain a 20 % w/v polymer solution. The polymer solution was added to deionized water in a 1:20 ratio to obtain a 1 % w/v polymersome dispersion and stirred in 12 mL-stirred tank reactors at 4000 rpm with an S-type stirrer for 1.25 h according to the method of Poschenrieder et al. [40 ]. The polydispersity index, the intensity-based diameter (z-average) and the number-based mean diameter (d_P) of the polymersomes were measured via dynamic light scattering on a ZetaSizer Nano-S (Malvern Instruments, Malvern, UK). For each polymersome preparation, the polydispersity index was ≤0.2 with a z-average of approximately 185 nm in a narrow, monomodal size distribution and a d_P of 110 nm. The average aggregation number has been determined to 43,000 polymer chains per polymersome [40 ]. Polymersome concentrations above 1 % w/v were obtained by concentrating the polymersome dispersion in 10 kDa Vivaspin 20 ultrafiltration units (Sartorius, Göttingen, Germany).

Wild-type HEK 293T cells were cotransfected with both the vector containing the desired hPLD3-sequence and the pCMV(CAT)T7-SB100(AL) vector (Dr M. Gossen) containing a Sleeping beauty (SB100) transposase gene. Two days after transfection, the cells were further cultivated in selection medium containing 7 μg/ml Puromycin (InvivoGen). Monoclonal cell lines were further generated by serial dilution in 96-well-plates. Clonal single-cell colonies were tested for expression efficiency using SDS-PAGE and western blot.
The obtained cell lines were grown to ∼90% confluency in T175 cell culture flasks (Sarstedt) under selection conditions for purification. Production medium (100 ml) containing only 2.5% fetal calf serum was added for 7 days and harvested by spinning down cell debris twice for 5 min at 500 g, followed by sterile filtration. The cell culture supernatant was concentrated to a final volume of 50 ml and purified on the AEKTA protein purification system using the HisTrap HP Ni-charged IMAC column (GE Healthcare) and subsequent size-exclusion chromatography using a Superdex 75 column (GE Healthcare) with PBS as the final solvent. The obtained fractions were pooled and concentrated by centrifugation in a Vivaspin 20 ultrafiltration unit (cutoff 10 kDa, Sartorius) for 20 min at 3300g and 4 °C. Purity was confirmed by SDS-PAGE and Coomassie stain.

Recombinant adeno-associated viruses were prepared as described previously (Burger and Nash, 2016 (link)). A mixture of plasmid DNAs including (1) AAV-DCX-Cre, pAdΔF6, and pAAV2/9, (2) AAV-CAG-DIO-EGFP, pAdΔF6, and pAAV2/9, or (3) AAV-CAG-DIO-mCherry, pAdΔF6, and pAAV2/9, was used to transfect AAVpro 293T cells (Takara Bio) seeded on twenty 15-cm dishes using polyethyleneimine “Max” (PEI-Max; Polysciences). After 2 d, cells were collected and lysed by three freeze–thaw treatments and lysates treated with Benzonase nuclease (Millipore). The rAAVs were purified from the lysates by ultracentrifugation using a discontinuous gradient of iodixanol (OptiPrep; Abbott Diagnostics Technologies AS). Banded rAAVs were collected from the 40% iodixanol layer, and the iodixanol buffer exchange to PBS and concentration of the rAAV was performed using a Vivaspin 20 ultrafiltration unit (100 kDa molecular weight cut off; Sartorius). For determination of the genomic titer, rAAVs were treated sequentially with DNaseI and Proteinase K, and the quantity of viral DNAs was determined by quantitative PCR using a StepOnePlus real-time PCR system (Applied Biosystems).

To enrich the secretome proteins, the supernatants from control and treated PACs (10 ml each) were concentrated separately to 2 ml with a Vivaspin 20 Ultrafiltration Unit (Sartorius Göttingen, Germany). The resulting samples were then subjected to protein precipitation to reduce the volume and enrich the proteins. The precipitation was carried out by adding 3 volumes of ice-cold acetone containing 10% methanol and incubating overnight at −20°C. Precipitated proteins were pelleted by centrifugation at 12,000 g for 45 min at 4°C. The pellets were dried and resolved in urea buffer (30 mM Tris-HCl pH 8.5, 9.5 M urea, 2% CHAPS). The protein concentration was determined according to the Bradford method using BSA as the calibrator.