Primary human islets

Gene editing of human primary islets

Human primary cadaveric islets were purchased from Prodo Labs and cultured overnight in PIM media (Prodo). The CRIPSR-Cas9 technology was used for the disruption of the B2M and CIITA genes. Islet clusters were dissociated in single cells using AccuMax (StemCell Technologies) for 10 min at 37°C. The following gRNA sequences were used for the human B2M gene 5’-CGTGAGTAAACCTGAATCTT-3’ and human CIITA gene 5’-GATATTGGCATAAGCCTCCC-3’. The Lonza P3 Primary Cell 4D-Nucleofector X Kit (V4XP-3032, Lonza) was used for the transfection of the islet cells. Briefly, cells were transduced with a final concentration of 50 million per ml in P3 buffer. Twenty microliters of the cell suspension were pipetted in one well of the 8-strip containing 13 mg Cas9 enzyme and 6.5 mg sgRNA, respectively. Lonza's 4D-Nucleofector was used for the electroporation with the preset program CA-137. Islet cells were transferred in U-bottom 96-well plates containing 50,000 cells per well in PIM(S) media (Prodo) and rested for 1 h at 37°C and 5% CO₂ before moving the plate on the belly dancer orbital shaker (IBI Scientific) for islet re-clustering. Complete media change was performed after 48 h, and islet clusters were incubated on the belly dancer for another 24 h. 

Islet clusters were dissociated again in single cells using AccuMax for cells sorting using the anti-HLA-A,B,C antibody (clone G46_2.6, BD Biosciences) or IgG1 isotype-matched control antibody (clone MOPC-21, BD Biosciences) and anti-HLA-DR,DP,DQ antibody (clone Tu3a, BD Biosciences) or IgG2a isotype-matched control antibody (clone G155–178, BD Biosciences). Double negative cells were sorted in the BD FACS Aria II and replated in U-bottom 96-well plates as described above for islet re-clustering on the belly dancer orbital shaker. After 24 h, islets were dissociated in single cells for CD47 and luciferase transduction with a CAG-CD47 LVV (custom order, Thermo Fisher) at a MOI of 5 and a CAG-luciferase LVV (custom order, GenTarget) at a MOI of 20. Spinfection was performed with the presence of 10 mg/ml protamine sulfate (Fresenius Kabi) at 300 g for 15 min. Cells were replated in U-bottom 96-well plates as described above for islet re-clustering on the belly dancer orbital shaker. After 48 h, cells were dissociated in single cells using AccuMax and underwent cell sorting for human CD47 with anti-CD47 antibody (clone B6H12, BD Biosciences) or IgG1 isotype-matched control antibody (clone MOPC-21, BD Biosciences) on BD FACS Aria II. Luciferase expression was confirmed by adding D-luciferin (Promega). Signals were quantified with AMI HT (Spectral Imaging) in maximum photons s⁻¹ cm⁻² sr⁻¹. Islet cells were replated in U-bottom 96-well plates as described above for islet re-clustering on the belly dancer orbital shaker until transplantation.