Three different studies were performed. In a first study, the biodistribution of intravenously administrated ePB-MSCs was evaluated in four dogs. In the second study, the biodistribution of intramuscularly and subcutaneously administrated ePB-MSCs was evaluated in four dogs. Finally, in a third study, the biodistribution of a higher dose of ePB-MSCs following intravenous administration was evaluated in three dogs.
The different animal studies (approval number EC: 2019_003 and 2019_006) and the blood collection of the donor horses (approval number: EC_2016_003) were approved by the ethics committee of Global Stem cell Technology. The ethics committee is approved by the Flemish government with permit LA1700607. The study was good clinical practice compliant (VICH GL9) and all animal handlings were conducted according to European, national and regional animal welfare regulations (Directive 2001/82/EC as amended, Belgian animal welfare legislation (KB 29/05/2013), Directive 2010/63/EU and EMEA/CVMP/816/00-Final).
Four healthy adult research dogs were included in the two first studies; three dogs from the second study were re-used for the third study. All dogs were purpose bred adult beagles (16-23 months). Two males and two females were included in the first two studies and one male and two female dogs were included in the third study. The dogs were housed in groups of 2, in a pen of 4 by 4 by 2 m (L x W x H) so permanent visual, olfactorial, tactile and auditive contact between dogs was possible. Toys were provided for the dogs to play with in their pen. Cleaning of the dog pens was performed daily. The floor was covered with wood shavings to improve lying comfort for the dogs. The animals were let out in dog runs to play and run for minimal 1 hour a day. After the study this minimal time was increased depending on the need of the dogs.
A daily general physical assessment was performed for each study evaluating the following parameters: rectal temperature, respiratory rate, heart rate, mucosal membranes, capillary refill time, body conditions score, mentation and hydration.
Control product preparation
For the preparation of the control product (CP), 20 ± 5 millicurie (mCi) (740 ± 185 megabecquerel (MBq)) of freshly eluted 99mTc Pertechnetate (99m Tc) from a molybdenum generator (GE health care, Eindhoven, The Netherlands) was added to 1 mL of Dulbecco’s Modified Eagle low glucose Medium (DMEM) (Life Technologies Europe BV, Belgium).
Collection and culture of ePB-MSCs
As previously described by our group , the ePB-MSCs were good manufacturing practices (GMP) manufactured in a GMP-certified site (number: BE/GMP/2018/123). Briefly, blood was taken from the jugular vein of a donor horse (approval number EC: EC_2012_001 and 2016_003) and the MSCs were isolated. The serum was analyzed for a range of transmittable diseases by Böse laboratory (Harsum, Germany). As already described by our group , the blood was centrifuged and the buffy coat was collected for gradient centrifugation. After washing, the ePB-MSCs were cultivated until passage 5 and a characterization for viability, morphology, presence of cell surface markers and population doubling time was performed. Next the ePB-MSCs were frozen as an intermediate cell stock. When characterization was completed, the intermediate cell stock was thawed and cultivated until passage 10 before being trypsinezed, resuspended, filtered twice trough a 40 µm filter and vialed at 3x105 cells/mL in a mixture of DMEM and 10% dimethylsulfoxide (DMSO). The vials were stored at -80°C until further use.
99m Tc-labelling of the ePB-MSCs
The technique of 99mTc labelling the ePB-MSCs was based on an optimization study recently described by our group (paper submitted). First, stannous chloride powder (Sigma Aldrich, US) was dissolved in sterile basic water (pH 8.5). Next, 0.9 x 106 ePB-MSCs were thawed in the hand palm, transferred into growth medium and centrifuged for pelleting. The cell pellet was then re-suspended in 4 mL of saline and mixed with 5 µg SnCl2 and 45 ± 5 mCi (1665 ± 185 MBq) of freshly eluted 99mTc from a molybdenum generator (GE health care, Eindhoven, The Netherlands). Next the preparation was incubated for 30 minutes at room temperature before being centrifuged. The cell pellet was washed with 5 mL DMEM and centrifuged again. The final cell pellet was resuspended in 1 mL of DMEM and the viability of the ePB-MSCs following the labelling was determined using trypan blue. The radioactivity of the supernatant was measured after each centrifugation step in a dosiscalibrator and used to calculate the labelling efficiency.
In the first study, each dog received two intravenous injections; first the dogs were injected with the control product: freshly eluted 99mTc dissolved in DMEM and at least 7 days later they received a second injection with 99mTc-labelled ePB-MSCs. For the second study, 4 injections were administered to each dog. The dogs first received an IM injection with the control product, next a SC with the control product, then an IM injection with the 99mTc-labelled ePB-MSCs and finally a SC administration of the 99mTc -labelled ePB-MSCs. At least 7 days separated each injection. In the third study, the three dogs received a single IV injection with 99mTc-labelled ePB-MSCs.
The dogs were put under general anesthesia and positioned in sternal recumbency on the gamma camera before each injection. To obtain general anesthesia, the dogs were first sedated with dexmedetomidine (12-25 µg/kg IM), next induction was obtained with propofol (dosage on effect) and anesthesia was maintained with isoflurane 1.2-1.4% (on effect) in 100% oxygen following endotracheal intubation. The intravenous injection was administered through a 22-gauge catheter in one of the cephalic veins, the intramuscular injection was performed in the left quadriceps muscle and the subcutaneous injection was administered at the back of the neck.
A two-headed gamma camera, equipped with low energy high resolution collimators (GCA 7200 A; Toshiba) was used for the scintigraphic investigation. The whole body scan was obtained with the detectors of the SPECT scanner moving simultaneous dorsally and ventrally from head to tail of the dog over 10 minutes. All dogs were kept under general anesthesia during all the acquisitions. For all 3 studies, data collection of the first hour consisted of 6 successive acquisitions of each 10 minutes. The start of the first acquisition was simultaneous with injection of the radioactive compound and the dog remained in the same position for all 6 scans. Next, in the first study, total body scans (each lasting 10 minutes) were performed at 2h, 4h, 8h, 12h, 24h and 36h after placebo control and labelled ePB-MSCs administration using propofol (dosage on effect). For the second and the third study, 6 successive 10 minutes’ total body scans performed during the first hour after injection were followed by total body scans (each lasting 10 minutes) at 6h and 24h after each injection. For all studies, a syringe with a known amount of radioactivity to calculate % injected dose (ID), was simultaneous scanned with the dog. Care was taken for the dog’s re-positioning on the table, to avoid too much spatial deviation on the scans following the first hour scans.
First, the distribution of the placebo control and the labelled ePB-MSCs was assessed descriptively through the whole body. Consequently, the radioactivity was quantified in different manually drawn regions of interest (ROI) on the dorsal and ventral view of the whole body scans (matrix size 512x1024) using the free-hand region of interest tool of a DICOM viewing software platform (Hermes MultiModalityTM, Nuclear Diagnostics, Sweden). A geometric mean of dorsal and ventral activity for each time point and each ROI was calculated to compensate for attenuation. Relative uptake was expressed as % of decay corrected injected activity for each region of interest per time point and calculated based on the known standard activity. To keep shape and sizes (number of pixels) of the different organ ROI’s uniform, a ROI template was created per study and per dog and used for the different time points. A specific organ ROI was drawn on the image on which the organ was best delineated and thereafter used for the other images. Due to minor positioning deviations in between scans, ROI’s had to be replaced on some images, however without changing the shape and size.
Due to the low sample size of four animals, only the overall effects in the heart, lung, liver and bladder following intravenous injection were taken into account for statistical analysis. The data were analyzed with SAS® statistical analysis software (version 9.4, SAS Institute Inc., Cary, NC, USA). For the intramuscular and subcutaneous injections no statistical analysis was performed since a high radioactivity uptake remained at the injection sites following the injections of the radiolabelled ePB-MSCs and only a descriptive evaluation seemed appropriate. The overall statistical difference between intravenous administration of the free 99mTc and the radiolabelled ePB-MSCs in the heart, lungs, liver and bladder was calculated using the area under the curve (AUC). The AUC was calculated using the trapezoidal method and can be written as a weighted sum of the observations. To allow a better interpretation of the AUC, it was presented as the weighted mean of the observations, using the weights of the observations in the AUC sum. A paired t-test was performed for this AUC for each organ separately, using the dog as a block effect. The time effects were described descriptively. The normality distribution assumption of the residuals was tested using the Shapiro-Wilks test and could not be rejected.