Nod scid il2rγ null mice

Tumor growth and spontaneous metastasis formation assayed by injecting tumor cells orthotopically into inguinal mammary fat pads (6- to 8-week-old female NOD/SCID/IL2Rγ-null mice) (Jackson Laboratory, Bar Harbor, ME, USA). Mice anesthetized with isoflurane, injected with 1.5 × 10⁵ cells in Hank's Balanced Salt Solution (Gibco); killed 6±0.5 weeks post injection; tumors dissected, weighed, flash frozen, stored (−80 °C) or fixed: 3.8% formaldehyde, imaged with a fluorescence microscope, and embedded in paraffin and sectioned. Lungs were collected, fixed: 3.8% formaldehyde, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). ZsGreen-positive foci were counted in left pulmonary lobe using ImageJ.

Human pseudoislets were transduced and cultured as described above. Batches of 300 pseudoislets were resuspended in cold Matrigel and transferred into the left renal capsular space of host animals using a glass micro-capillary tube. Transplant recipients were 8-week-old male NOD scid IL2Rγnull mice (stock number 005557; The Jackson Laboratory) and were anesthetized using ketamine/xylazine. Appropriate depth of anesthesia was confirmed by lack of toe-pinch response. One-month post-transplantation, mice were administered an intraperitoneal glucose injection at a dosage of 3 g/kg body weight. Glucose measurements and blood samples were collected via the tail vein at 0, 15, 30, 45, 60, 120, and 180 min post glucose injection. Human insulin is distinguishable from mice insulin, which allowed us to measure its levels by a human insulin ELISA kit (Mercodia).

All mice were maintained under specific pathogen-free conditions and experimental procedures were performed according to the institutional animal welfare guidelines approved by Institutional Animal Care and Use Committee for the University of Kansas Medical Center.
For subcutaneous tumour growth assays, cells were dissociated into single-cell suspensions using non-enzymatic cell dissociation solution (Sigma Biochemicals) and numbers of live cells were counted following trypan blue staining (Thermo Fisher Scientific). Cell suspension in 50 μl of 4.5 mg ml⁻¹ Matrigel (BD Biosciences) in Hank's balanced salt solution was subcutaneously injected into flanks of NIH-III nude mice (Charles River). Tumours were measured three dimensionally two to three times a week for 17–20 days. For tail vein assays, 150 μl of cell suspension (5 × 10⁴) was injected into the lateral veins of nude mice. Mice were monitored for laboured breathing and the numbers of pulmonary tumour nodules were evaluated 6 weeks after injections. For orthotopic injections, 15 μl of cell suspension (1 × 10⁵) was injected into femoral bone marrow space of anaesthetized NOD-scid IL2Rγ^null mice (The Jackson Laboratories)22 (link). When the tumours reached ∼1 cm in thigh diameter, the mice were killed. The weight of the primary tumours and numbers of tumour nodules in the lungs and liver (>0.5 mm) were measured.