Isolation of mesenchymal stem cells
Ethical approval for the collection of bone marrow aspirates and blood was received from the Ethics and Welfare Committee at the Royal Veterinary College (URN 2013 1230R 2005). No horses were euthanased for the sole purpose of obtaining tissues for this study. Bone marrow-derived MSCs (n = 3) were obtained and expanded as described previously [9] and P0 to P2 passage cells were stored in Bambanker™ cell freezing medium (Anachem, Luton, UK) in liquid nitrogen until use. For experiments, cells were seeded in D10 medium (Dulbecco’s modified Eagle’s medium, supplemented with foetal calf serum (10% v/v), 100 U/ml penicillin, and 100 U/ml streptomycin; all from Invitrogen, Paisley, UK) and were expanded to the required numbers. Cells were then detached from the culture flasks by trypsin–ethylenediamine tetraacetic acid (Sigma-Aldrich, Gillingham, UK) for storage media and needle gauge experiments.
Effect of storage media
MSCs (2.5 × 106 cells) were resuspended in the test media at a concentration of 5 × 106 cells/ml and transferred to 1 ml cryotube vials (PAA, Yeovil, UK). Resuspension media were as follows: D10; allogenic BMA; hyaluronic acid (as sodium hyaluronate at a concentration of 10 mg/ml in isotonic sodium chloride–phosphate buffer, pH 7.0; Bayer PLC, Newbury, UK); allogenic plasma (heparinised) and allogenic serum obtained by routine venipuncture from a horse free of systemic disease; isotonic saline (Dechra Pharmaceuticals, Northwich, UK); allogenic platelet-rich plasma (prepared by routine venipuncture into 3.8% trisodium citrate (ratio 9:1), after which platelets were concentrated by centrifugation as previously described [20] and the platelet pellet was resuspended in D10 (2 ml for platelets pellets prepared from 20 ml plasma)); and cryogenic cell freezing medium (90% equine serum, 10% dimethyl sulphoxide).
Vials were stored at 4 to 8°C until analysis except for those containing cell-freezing medium, which were stored in dry ice (-78°C). Prior to assaying, these vials were warmed briefly in a 37°C water bath with care to avoid heating beyond melting point. Cells were thawed by slow addition (over 90 seconds) of an equal volume of lactated Ringer’s solution (Dechra Pharmaceuticals) to the vial. After storage for 12, 24, 48 and 72 hours, cells for all media were assayed for viability and cell proliferation as detailed below.
Effect of needle gauge
MSCs were diluted to a final suspension density of 5 × 106 cells/ml in D10. One millilitre of cell suspension from each cell line was set aside as a non-injected control group. One-millilitre aliquots of MSCs were slowly aspirated into a 2 ml syringe with the appropriate test needle attached (as is the practice for clinical MSC injection). Needle sizes of 21G (currently used for MSC injection), 23G (one gauge smaller) and 19G (one gauge larger) were used. All needles were 50 mm in length (Terumo Ltd, Bagshot, UK). The loaded 2 ml syringe was then fixed concentrically within a 30 ml syringe to enable mounting into a Flo-Gard GSP syringe pump (Baxter, Newbury, UK). The extrusion rate was set to 900 ml/hour and the volume (Volume to be Infused) set at 7.5 ml, which was appropriate to deliver a 1 ml volume from the 2 ml syringe over 30 seconds to mimic the slow, steady injection of MSCs applied in clinical practice. Cell viability, proliferation and apoptosis assays were performed immediately after extrusion of cells from the needles and on the non-injected control cells.
Viability assay
The viability of cell suspensions from both the storage media and needle experiments were determined by mixing 100 μl cell suspension with 100 μl of 0.4% trypan blue solution (Sigma-Aldrich) for 2 minutes. Cells were counted using a haemocytometer microchamber under a light microscope [21].
Cell proliferation assay
To assess the ability of the cells to proliferate following storage in the various storage media, a proliferation assay was performed using the alamarBlue® assay (AbD Serotec, Kidlington, UK). Then 5 × 103 cells were seeded in duplicate into each well of a microtitre plate in D10 and allowed to adhere for 24 hours. The media were replaced with 1 ml D10 containing 100 μl alamarBlue® reagent and cells were incubated for a further 4 hours, shielded from light. Then 100 μl aliquots were transferred to a black fluoro-microtitre plate (SPL LifeSciences, Singapore, UK) and fluorescence was measured at 570 nm (excitation) and 585 nm (emission) (Infinite M200 PRO fluorometer; Tecan, UK). The media in the cells was replaced with fresh D10 and the assay was repeated at 24, 48 and 72 hours as above.
Mesenchymal stem cell characterisation
Chondrogenic, adipogenic and osteogenic differentiation assays were performed and assessed as described previously [22].
Metabolic activity
Cell suspensions from the needle gauge experiments were assayed for metabolic activity using the alamarBlue® reagent. Cells were diluted to a concentration of 2.5 × 105 cells/ml in D10, and 100 μl alamarBlue® reagent was added and incubated at 37°C. At 4, 6, 8 and 24 hours, 100 μl aliquots of sample were then measured for fluorescence as detailed above. Readings obtained for needle injected cells were compared with values for the non-injected control cells.
Apoptosis assay
The Annexin V–fluorescein isothiocyanate enzyme-linked immunosorbent assay (Cayman Chemical, Ann Arbor, MI, USA) was used to assess the effect of needle size on induction of apoptosis. The assay was validated in preliminary assays for cross-reactivity of the Annexin V antibody to the equine antigen using positive inducers of apoptosis. Cells were treated with staurosporine (500 and 100 nM) and incubated at 37°C for 3 hours or with sodium azide (20 nM) and incubated at 37°C for 2 hours. Cells were also heated to 45°C for 10 minutes to induce apoptosis. In addition, test cells were included where 1 ml cells in a 2 ml syringe were extruded through a 21G needle, and non-injected cells were suspended in D10 or in fresh allogeneic plasma. The assay was performed according to the manufacturer’s instructions. Briefly, staining solutions were prepared in Annexin V binding buffer (0.01 M HEPES, pH 7.4; 0.14 M NaCl; 2.5 mM CaCl2) to contain either Annexin V–fluorescein isothiocyanate antibody or propidium iodide, and were protected from direct light. Samples (100 μl containing 500,000 cells) were washed twice in 1 ml Annexin V binding buffer by centrifugation at 350 × g and cells were resuspended by gentle agitation in 250 μl of each staining solution. Samples were wrapped in aluminium foil to protect from direct light and incubated for 10 minutes at room temperature. Cells were washed as before and resuspended in 100 μl Annexin V binding buffer, and 10 μl aliquots were applied to a glass slide and mounted under a coverslip to visualise by fluorescence microscopy (using an Olympus BX61). The number of cells staining positive for propidium iodide (dead cells) using a rhodamine filter (excitation 540 nm and emission 570 nm) or Annexin V (cells in early apoptosis) using a fluorescein filter (excitation 485 nm and emission 535 nm) were counted. Means of cell counts from six fields of view per slide were determined at 20× magnification for both stains and the total cell number under a bright field. Test groups were analysed at 0, 2, 4 and 24 hours post injection.
The validation experiments confirmed that the Annexin V antibody (anti-human antibody) cross-reacted with the equine antigen and revealed that the strongest inducers of apoptosis were 500 nM staurosporine and injection through the 19G needle. While all conditions induced apoptosis, there was no significant difference between any of the known apoptosis inducers. Comparison of the percentage of Annexin V immunofluorescent cells with the inducers of apoptosis with that of non-injected control cells in either D10 or fresh plasma revealed a significant increase in the proportion of early apoptotic cells with all apoptotic stimuli applied (P < 0.01; Figure 1).There was no significant difference in the mean total cell number between test groups, enabling confident comparison of the number of Annexin V-positive and propidium iodide-positive cells calculated as a percentage of total cells.
Statistical analysis
Where descriptive analysis indicated that the data were not normally distributed, a log transformation was performed to normalise the data prior to analysis. Longitudinal data were analysed using a mixed-effects linear regression model in PASW (version 18; IBM, Portsmouth, UK), with Sidak correction. In order to interrogate the relationship between and within the multiple factors on cell viability or proliferation, statistical analysis was performed using analysis of variance within a mixed-effects model. P ≤0.05 was considered significant.