## 3 Demonstration of the Steps Based on the hypACT Inject Auto Device
### Maintaining Mesenchymal Stem Cell (MSC) culture  
Cell culture procedures were carried out in a laminar flow tissue culture hood. Bone marrow-derived mesenchymal stem cells (MSCs; ATCC, Manassas, VA, USA) were cultured in T-75 flasks in an incubator at 37 \({}^{\circ}\)C, 5% CO\({}_{2}\) and 95% humidity. MSCs were maintained in stem cell medium: Dulbecco's modified Eagle's medium containing 4.5 g/L glucose and L-glutamine (Lonza, Basel, Switzerland) supplemented with 10% foetal bovine serum (FBS; EuroClone, Pero, Italy), 1% Penicillin-Streptomycin (Sigma-Aldrich, St. Louis, MO, USA) and 0.75 ng/mL basic fibroblast growth factor (Sigma-Aldrich, St. Louis, MO, USA). The culture medium was refreshed three times a week. 5.2 Preparation of different supplementation containing media  
As positive control stem cell medium (FBS medium), as negative controls serum-free medium and human serum albumin (Behring, King of Prussia, Pennsylvania, USA)-containing medium (HSA medium) were used. Phlebotomy occurred under ethical approval (IRB approval number: 33106-1/2016/EKU, 12.07.2016) from healthy donors, men and women, aged 24-45 years. SPRF was isolated using the device as shown in Figure 2 (device SPRF). The device SPRF contains a small amount of red blood cells, which may be harmful to cell viability. They can be eliminated by another centrifugation at 1700 g for 8 min after isolation (twice centrifuged device SPRF). The reference SPRF was isolated manually. Fifty millilitres of blood was drawn using an ordinary syringe and the blood was poured into a centrifuge tube, containing 10 g glass beads as glass surface to promote blood clotting. It was immediately centrifuged at 1700 g for 8 min, while the fibrin matrix was formed. To squeeze out SPRF from the fibrin matrix, it was centrifuged again at 1700 g. Each cell culture media contained 10% supplementation, except for serum-free and HSA medium, where the amount of HSA was normalized to the protein content of SPRF. 5.3 Comparing the effect of different serum containing media on MSC cell viability  
MSCs (4 passages) were seeded onto 24 well plates at a density of 1500 cells/well in 500 \(\mu\)L stem cell medium and the culture medium was changed on the first day to the different supplementation containing media. The medium was changed every two days. Cell viability was measured on the sixth day using the Cell Proliferation Kit II (XTT; Roche, Mannheim, Germany) according to the manufacturer's instructions. Absorbance was measured by a microplate spectrophotometer (Biotek, Winooski, Vermont, USA) at 480 nm with a reference wavelength at 650 nm. The cells were visualized by Live/Dead staining. MSCs were washed with PBS and stained in PBS, containing 1 \(\upmu\)M Calcein-AM (Invitrogen, Carlsbad, CA, USA), 4 \(\upmu\)g/mL ethidium homodimer (Invitrogen, Carlsbad, CA, USA) and 20 \(\upmu\)g/mL Hoechst (Invitrogen, Carlsbad, CA, USA) for 20 min, then washed again in PBS. Images were taken by an inverse fluorescent Nikon microscope. * Results The results of XTT measurement (Figure 3) do not show any significant differences between device SPRF, twice centrifuged device SPRF and glass bead SPRF. One-way analysis of variance (ANOVA) with [PERSON]'s post hoc test was performed to compare the means of groups using Prism 7 software. The significance level was \(p<0.05\) and data are presented as mean \(\pm\) SEM (n = 4). The results of microscopic imaging show (Figure 4) that MSCs cultured in the three kinds of SPRF media reached roughly similar density in the wells and they have the same MSC-like shape. * Evaluation The main conclusion of the clinical evaluation was that the blood separation product prepared by our device is at least as safe to use as a PRF isolation method that is already used in the practice, thus that is a way to claim clinical safety of the device. However, the optimal path to investigate clinical safety of medical devices needs to be evaluated on a case-by-case basis; nevertheless, the present path was applicable for the device.