Allogenic Adipose Derived Mesenchymal Stem Cells and Vitamin D Supplementation in Patients With Recent-onset Type 1 Diabetes Mellitus

Adipose tissue samples will be obtain through liposuction procedures of three healthy volunteers undergoing aesthetic surgery at Clementino Fraga Filho (the hospital of Federal University of Rio de Janeiro, Brazil). Donors´sorology wil have to be negative for syphilis, Chagas disease, Hepatitis B virus, Hepatitis C, HIV 1 and 2, and Cytomegalovirus.

The adipose tissue-derived stem/stromal cells (ASCs) that wil be extract on healthy volunteers will be isolate, culture and samples will be process at Core Cell Technology of Pontifícia Universidade Católica do Paraná. Briefly, the procedures are:

1. 100 ml of adipose tissue will be wash in sterile phosphate-buffered saline (Gibco Invitrogen). A one-step digestion by 1 mg/ml collagenase type I (Invitrogen) will be performe for 30 minutes at 37°C during permanent shaking, follow by filtration step through a 100 µm mesh filter (BD FALCON, BD Biosciences Discovery Labware, Bedford, MA, USA). The cell suspension will be centrifuge at 800 g for 10 minutes, and erythrocytes were removed by lysis buffer, pH 7.3.
2. The remaining cells will be wash at 400 g for 10 minutes and then culture at a density of 1×105 cells/cm2 in T75 culture flasks and DMEM-F12 (Gibco Invitrogen) supplemente with 10% of fetal calf serum, penicillin (100 units/ml) and streptomycin (100 μg/ml). The culture medium will be replace three days after seeding, and then twice a week.
3. ASCs will be subculture after reaching 80% confluence, with 0.5% trypsin/EDTA (Invitrogen) solution. Cells will be replate at a density of 4x103 cells/cm2 for expansion11A.
4. Quality control of cell suspension sterility will be evaluate by tests to detect bacteria and fungi (Bact / Alert 3D, Biomerieux), endotoxins (Endosafe ™ PTS, Charles River) and Mycoplasma (KIT MycoAlert ™ PLUS Mycoplasma Detection, Lonza). Cell viability will be performe by flow cytometry using the vital dye 7-AAD (7-Aminoactinomycin D - BD#559925) to determine the percentage of viable cells and Annexin V protein (BD#51-65875X) to determine the percentage of cells in apoptosis. Cytogenetic analysis wil be performe by GTG-banding method.
5. Cells will be phenotypically characterize by flow cytometry before the clinical application, using the following monoclonal antibodies: FITC-labeled CD14 (BD#555397), CD45 (BD#555482), CD19 (BD#555412), CD44 (BD#555478); PE-labeled CD73 (BD#550257), CD90 (BD#555596), CD166 (BD#559263), PerCP-labeled HLA-DR (BD#551375); APC-labeled CD34 (BD#555824), CD105 (BD#562408), CD29 (BD#559883) all purchased from BD (Pharmingen). At least 100.000 events wil bel acquire on a BD FACSCalibur™ flow cytometer (BD Biosciences), and data wil be analyzed using FlowJo 10 (TreeStar) software11A.

After the ASCs extraction, isolate, culture and process, the infusion in patients with recent-onset type 1 diabetes wil be according as describe bellow:

1. At the day of infusion, ASCs monolayer wil be dissociate as described above and 1x106 cells/kg of the recipient patient wil be ressuspend in 5 ml of saline solution with 50% albumin and 5% anticoagulant citrate dextrose solution. Cell suspension wil send to the hospital in a cooler with recycled ice.
2. Patients that wil receive ASCs wil be admitte to the hospital in the day of the infusion and wil discharge 24 hours after infusion. A single dose of ASCs wil be infuse in a peripheral upper arm vein during 15-20 minutes. Patients wil start takking oral cholecalciferol 2000 UI one day after the ASCs infusion.