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Supplementary information
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Methods
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MSC production
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Expanded MSC from bone marrow are classified as Advanced Therapy Medicinal Products and
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manufactured in the GMP-licensed Cell Therapy Facility of the UMC Utrecht. The bone marrow
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aspirates were obtained from third party non-HLA matched healthy donors as approved by the
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Dutch Central Committee on Research Involving Human Subjects (CCMO, Biobanking bone
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marrow for MSC expansion, NL41015.041.12). Either the bone marrow donor or the parent or
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legal guardian of the donor signed the informed consent approved by the CCMO. Bone marrow
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was separated using a density gradient centrifugation (Lymphoprep, Axis Shield, Oslo, Norway).
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MSC are isolated by plastic adherence and expanded using the MC3 systems and α-MEM
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(Minimal Essential medium) with L-glutamine from Macopharma (Tourcoing, France). In short,
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100-300 x 106 mononuclear cells in culture medium (α-MEM, 5% platelet lysate and 3.3 IU/ml
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Heparin) are seeded in a 2-layer CellStack (2-CS) using the seeding set. After 7 days, a medium
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exchange was performed using the exchange sets, depleting all nonadherent cells. When 80 –
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100% confluency was reached (± 10 days), the cells are harvested using trypsin (TrypLE™
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Select Enzyme™, Life technologies Corp. Grand Island, NY, USA). The Passage 1 (P1) cells
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were seeded in CellStacks (2-5 x 106MSC/2-CS), medium was exchanged and the cells were
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passaged after ± 6 days (P2). This procedure is repeated to obtain P3 cells for infusion. The mean
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number of MSC harvested at P3 was 59 x 106 MSC/2-CS. P3 cells were cryopreserved in MACO
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Biotech freezing EVA bags (Macopharma, Tourcoing,France) in 20 ml 0.9% Sodium Chloride
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(Fresenius Kabi, Bad Homburg, Germany); 10% CryoSure-DMSO (WAK-Chemie Medical
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GmbH, Steinbach, Germany); 5% Human Serum Albumin(Cealb, Sanquin, Amsterdam, the
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Netherlands). P3 cells were cryopreserved using a computer-controlled rate freezer (IceCube
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1810, Sy-Lab GmbH, Neupurkersdorf, Austria) in clinical cell dosages ranging from 20 – 200
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x106 MSC/bag and stored in the vapor phase of liquid nitrogen (< -150 °C)(1). The MSC batches
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were released if they fulfilled the following release criteria: immunophenotype of the MSC:
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>70% CD73+ cells , > 70% CD105+ cells, and >70% CD90+ cells, and <10% CD45+ cells and
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<1% CD3+ cells; sterility tests according to the European Pharmacopeia: negative for aerobic
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and anaerobe bacteria, fungi and yeast; mycoplasma < 10 CFU/ml and endotoxin < 1 IU/ml (<5
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IU/kg/hr). All MSC batches fulfilled the criteria as described by Dominici et al (1) and all MSC
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were >95 % positive for CD 73/CD 90/CD 105. The MSC bags were thawed in a water bath at
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37 °C, kept on ice while a sample was taken for cell counting and infused intravenously within 1
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hour at the clinical wards. The ability of thawed GMP-grade MSC to suppress T-cell
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proliferation was assessed during the implementation of the MSC production from 5 different
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donors. Different MSC:Peripheral Blood Mononuclear Cells (PBMC) ratios were assessed as
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shown in supplementary figure 1. T-cell proliferation was decreased to 38% (mean of all 5
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assessed MSC donors when added in ratio MSC: PBMC of 1:1). Cell viability of thawed MSC
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was assessed for each product throughout the whole study and the median viability was 95%
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(mean 93,5%, standard deviation 7,6%).
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Assessments
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FACS stainings were performed with: CD3-PerCP, CD4-PerCP, γδ-TCR-APC, CD80-APC-H7,
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CD86-PE-Cy7 (BD Pharmingen), CD4-PE, CD8-PerCP, CD19-PerCP, CD14-PerCP, CD141-PE
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(BDCA3) (BioLegend), CD3-eFluor-450, CD4-PE-Cy7, CD4-Alexa Fluor-780, CD8-APC,
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CD25-FITC, CD127-PE-Cy7, HLA-DR-FITC, FoxP3-APC, CD20-PacBlue (eBioscience),
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CD1c-APC (BDCA1), CD303-FITC (Miltenyi). Samples were analyzed with an LSR-II flow
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cytometer (BD Biosciences) and acquired data were analyzed using FACS Diva software (BD
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Biosciences).
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Reference List
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(1) Dominici M, Le BK, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria
for defining multipotent mesenchymal stromal cells. The International Society for Cellular
Therapy position statement. Cytotherapy 2006;8(4):315-7.
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