T ime-related decline of human islet allograft (TX) function in generally immunosuppressed type 1 diabetic patients (1-5) has led us, after years of preclinical study (6), to initiate a phase 1 pilot clinical trial of microencapsulated TX into 10 nonimmunosuppressed patients with type 1 diabetes, under permission and surveillance by the Italian Ministry of Health (file no. 19382, PRE 805, 5 September 2003).
RESEARCH DESIGN AND METHODS
Human islet procurementHuman islets were isolated from singledonor pancreases according to the Edmonton protocol (1). Only preparations complying with standard quality control criteria (1) were considered for TX. The islets were cultured for 24 h in HAM F12 (Celbio, Milano, Italy), supplemented with antibiotics and 1.25% human albumin (Kedrion Spa, Milano, Italy) at 37°C in 95% air/CO 2 .
MicroencapsulationThe islets were washed and thoroughly mixed with 1.6% endotoxin-and pyrogenfree sodium alginate (Stern Italia, Milan, Italy) that had been highly purified according to U.S. Pharmacopeia. Upon extrusion through a microdroplet generator (droplets generated by combination of air shears and mechanical pressure by a peristaltic pump), microdroplets containing the islets, upon collection in a CaCl 2 bath, turned into gel microbeads. The beads, containing 1-2 islets and measuring an average 500 m in diameter, were sequentially double-coated with 0.12% and 0.06% poly-L-ornithine (Sigma) and finally with 0.04% sodium alginate (Stern) (7).
Patient recruitmentTen patients with long-standing type 1 diabetes on intensive insulin therapy regimens (lispro prebreakfast, lunch, and supper and glargine presupper) and undetectable serum C-peptide responses (sCPRs) were tested for complete blood chemistry, parameters of diabetes control (GHb, daily blood glucose profiles, and insulin requirement), anti-GAD 65 antibodies, and islet cell antibodies. Chest Xray and abdominal echography were also performed.
Pre-and post-TX patient assessmentThe patients were maintained on euglycemia overnight before TX by supplementing exogenous insulin as required. After TX, the patients were monitored hourly for blood glucose (range: 80 -150 mg/dl) and insulin requirement. sCPR samples, assayed by radioimmunoassay (sCPR sensitivity: 0.06 ng/ml [intra-assay CV 3.7-4.5%, interassay CV 4.4 -5%]) (TechnoGenetics, Milan, Italy) in our laboratory, were drawn twice daily for the first 7 days and weekly thereafter, both in basal and 90 min after meals. A 75-g oral glucose tolerance test (OGTT) was scheduled for patient 1 at 60 days post-TX. At 7 days post-TX, the patients were discharged and instructed to continue blood glucose home selfmonitoring, with adjustments of insulin requirement. At 30 days post-TX, control abdominal echography was scheduled.
Encapsulated human islets for TXA total of 400,000 and 600,000 microencapsulated islets (islet equivalents normalized to 150 ) were grafted in patient 1 and patient 2, respectively. The final TX volume included 50 ml of microcapsules diluted in 100 ml of saline. Empty microcaps...
