Ficoll paque solution

MSCs were isolated from the peripheral blood (PB) of six sheep that were not part of the wound model experimental design. From each animal, 100 ml of blood was taken from the jugular vein using a vacutainer containing the anticoagulant Li-heparin. The mononuclear cells were isolated using the protocol of Martinello et al. [13 (link)]. Briefly, the blood was diluted 1:1 with PBS (phosphate-buffered saline) and placed on Ficoll-paque solution (Amersham Biosciences) to obtain mononuclear cells in interphase after centrifugation. Cultures were maintained at 37 °C with 5% CO₂ in growth medium (DMEM 5671, Sigma-Aldrich) with 10% FCS (fetal bovine serum, Euroclone). On the day of application, PB-MSCs were trypsinized with 0.25% trypsin-EDTA (Euroclone, Italy) and resuspended in hyaluronic acid (Hyalgan®, Fidia).

Blood samples were obtained from both patients diagnosed with psoriasis (n = 5) and healthy volunteers (n = 5), all of whom provided written informed consent to participate in this study. Approval for this study was granted by the Institutional Review Board of Hallym University Kangnam Sacred Heart Hospital (IRB No. 2022-03-022). Peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples of both patients with psoriasis and healthy volunteers. A 1:1 ratio of human blood samples to Ficoll-Paque solution (1.077 g/mL; Amersham Biosciences, Uppsala, Sweden) was used, followed by centrifugation at 800 × g for 15 min. The cells from the interphase were subsequently washed three times with PBS containing 5 mM EDTA. PBMCs derived from patients with psoriasis were treated with Benzo[a]pyrene (BaP) at a concentration of 10 µM (Sigma‒Aldrich, St. Louis, MO, United States) for 24 h. Subsequently, exosomes were isolated from BaP-treated PBMCs of patients with psoriasis, as well as from non-treated PBMCs of patients with psoriasis. Additionally, PBMCs from healthy controls (HCs) were treated with 20 μg/mL exosomes for 24 h.

MSCs were isolated from three sheep that were not involved in the current experimentation. 100 mL of peripheral blood were collected from the jugular vein of each animal using a vacutainer coated with Li-heparin (Becton, Dickinson and Company, Franklin Lakes, New Jersey, United States), in order to avoid the formation of blood clots. MSCs were isolated following the protocol described by Martinello et al. (19 (link)). In brief, the blood was diluted 1:1 with phosphate buffered saline (PBS) solution and added onto a Ficoll-paque solution (Amersham Biosciences, Little Chalfont, United Kingdom) to obtain mononuclear cells (including MSCs) in the interphase after a density gradient centrifugation. Next, MSCs were cultured in a complete medium constituted of DMEM High Glucose (SigmaAldrich, St. Louis, MO, United States) supplemented with fetal bovine serum (FBS, 10%) (Euroclone, Milano, Italy) and antibiotics (1% penicillin–streptomycin; Euroclone, Milano, Italy). Cells were kept in an incubator at 37°C with 5% CO2. On the day of application, cells were detached from the tissue culture flasks using trypsin–EDTA 0.05% (Euroclone, Milano, Italy), counted, and diluted in PRP for the therapeutic application. Cell viability was checked along with counting using the trypan blue dye; all cells showed a viability ≥95%.

The isolation of Peripheral Blood Mononuclear Cells (PBMCs) was carried out on Ficoll–Paque solution (Amersham Biosciences AB Björkgatan 30, SE-751 84 Uppsala, Sweden) through density gradient centrifugation following the standard procedure of the manufacturer. According to the manufacturer’s instructions, total RNA was extracted from PBMCs by RNX™-Plus reagent (SinaClon, Tehran, Iran). The removal of genomic DNA was done by treating the extracted RNA with DNase I (Fermentas, Lithuania) at 37°C for 15 min, followed by verification through spectrophotometry and 1% agarose gel electrophoresis.

This study was approved by Héma-Québec’s Research Ethics Committee (CER#2020-010), and all participants signed an informed consent form. Whole blood (450 mL) was collected using the Leukotrap^® WB system (Haemonetics, Braintree, MA, USA) according to the manufacturer’s instructions. Immediately after the blood donation, PBMCs were isolated using gradient centrifugation with a Ficoll-Paque solution (Cytiva, Vancouver, BC, Canada) and Leucosep tubes (Greiner Bio-One; Monroe, NC, USA) according to the manufacturer’s instructions. PBMCs were collected and washed with DPBS (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 0.25% human albumin (CLS Behring, Ottawa, ON, Canada). The PBMCs were then suspended in a Plasma-Lyte solution (Baxter, Mississauga, ON, Canada) containing 5% human albumin and 18% CryoSure-Dex40 (WAK-Chemi medical, Steinbach, Germany), aliquoted, and frozen in liquid nitrogen for subsequent use.

We enrolled 20 healthy volunteers that were free of infections for at least four weeks prior to recruitment. Up to 70 mL of blood was drawn after informed consent and directly processed. We isolated peripheral blood mononuclear cells (PBMCs) using a density gradient centrifugation protocol (Ficoll Paque solution, GE Healthcare Bio Science AB, Uppsala Sweden). Isolated cells were resuspended in full RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany) and 100 U/mL penicillin plus 100 μg/mL streptomycin (both Invitrogen) and held at 37 °C in a humidified atmosphere containing 5% CO₂. For stimulation with lipopolysaccharide (LPS), cells were seeded into a 24-well plate. With the exception of control cells, all wells were stimulated with 1 μg/mL LPS for multiple periods of time (0 h/control, 0.5 h, 2 h, 4 h, 6 h, 24 h and 48 h).