In vitro assay

Bone marrow cells, consisting of various cell types including mesenchymal stem cells and fibroblasts, were cultured from adult male rats (Rattus norvegicus, SD strain). These cells were passaged from a growing primary stock in a T75 tissue culture flask (Sarstedt) into a 48 well tissue culture plate (IWAKI), with a cell density of 1.5 × 10⁵/ml. The cell culture media, henceforth referred to as ‘supplemented α-MEMS,’ comprised α-MEMS media supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin (all Life Technologies, Carlsbad, CA, USA). On the day of material addition to the cells, the supplemented α-MEMS was gently removed and fresh supplemented α-MEMS added, with 10% test material by volume, untreated controls receiving just fresh supplemented α-MEMS. All samples were filter sterilised using a Millex-GV 0.22 μM syringe filter unit (Merck, Kenilworth, NJ, USA). The cells were imaged for 24 h using an incubated Zeiss Axiovert 135M microscope with a Prior Proscan II motorised stage, at 37 °C with a 5% CO₂ in air feed. Each well of the 48-well tissue culture plate was then imaged for 24 h every 5 min using Metamorph software (MDS Analytical Technologies, Wokingham, UK), with each well having four fields of view. The viability and cell numbers were reviewed by analysing the images captured over the 24 h period and cell counts were taken at selected time points using the Metamorph software. The experiments, including cell counting, were ‘blind,’ the experimenter only being apprised of sample identity after data analysis and preliminary reporting. Test materials were rat PC12 conditioned medium and aqueous extract of rat hypothalamus, with and without anion exchange chromatography and with and without immunodepletion involving the anti-EPL001 rabbit antiserum. To prepare PC-12 conditioned media, 5 × 10⁵ PC12 cells were seeded onto T25 flasks and grown for three days (unless otherwise stipulated) to a density of 3 × 10⁶, in RPMI 1640, 2 mM Glutamine, 2% Foetal Bovine Serum, and the media harvested. PC12 conditioned media was subjected to anion exchange chromatography as follows: a total of 50 ml of conditioned media was diluted five-fold with 20 mM phosphate buffer and applied to a 5 ml Hitrap Q HP column (GE Healthcare 17-1154-01), pre-equilibrated in the same buffer, and eluted in 20 column volumes with a 0–1 M NaCl gradient using an AKTA FPLC (GE Healthcare, Little Chalfont, UK). Relevant biological activity had been seen in early studies involving sheep material (serum, follicular fluid) in <30 kDa fractions eluting at 0.2 M NaCl (tissue shrinkage in vivo (Hart, 2003)) and 0.8 M NaCl (reduced tumour cell proliferation in vitro (Hart, 2013, Example 7a therein)), directing attention at these anionic exchange fractions in particular. The hypothalamic extract was prepared using hypothalami from 50 adult male rats (Rattus norvegicus, CD strain), with tissue preparation at source (Charles River, Margate, UK), from animals maintained in compliance with all relevant national and international regulations in high quality barrier facilities. The hypothalami were homogenised from frozen and suspended in 10 ml of 50 mM Tris pH 8.0 with Complete Protease Inhibitor Cocktail (Sigma Aldrich, Dorset, UK). This was centrifuged at 20,000g for 1 h, the supernatant retained, and the sedimentation step repeated. This extract, neat and after anion exchange chromatography (as above) was submitted for western blot analysis. For immunodepletion studies an additional 50 ml of PC12-conditioned media was produced and incubated overnight with 200 μl anti-EPL001 antiserum at 4 °C. Antibody was then removed using a protein G column (Pierce, Fischer Scientific, Loughborough, UK). In every case immunodepletions were carried out on 0.7 ml of sample with the addition of a preabsorption step whereby 50 μl of equilibrated protein G beads were added for 1 h with gentle agitation to remove endogenous immunoglobulin. EPL001 peptide was provided for preabsorption at 100 μM. The work was conducted under UK Home Office Project Licence PPL 30/2280 and Personal Licence 30/353 and while specific ethical approval was not required the research was performed in accordance with the guidelines of the Ethical Review Board of the University of Reading, UK, and within its purview.