Edward H. Finke

Automated Method for Isolation of Human Pancreatic Islets

We describe an automated method for the isolation of human pancreatic islets. The procedure meets the following requirements: 1) minimal traumatic action on the islets, 2) continuous digestion in which the islets that are progressively liberated can be saved from further enzymatic action, 3) minimal human intervention in the digestion process, and 4) high yield and purity of the isolated islets. After purification on Ficoll gradients, an average of 164,600 islets/pancreas was obtained (2279 islets/g), with an average purity of 78.5% islets. The average volume and average insulin content of the final islet preparation were 348 mm3 and 93.4 U, respectively. The islets were morphologically intact with a normal degree of β-granulation and responded to glucose stimulation with a fivefold increase of insulin secretion over basal levels. The procedure is now being used for the initiation of the second phase of clinical trials on human islet transplants.

A Method for the Mass Isolation of Islets From the Adult Pig Pancreas

A method for mass isolation of islets from the pig pancreas is described. The procedure involves the use of an enzymatic and mechanical pancreatic digestion procedure followed by filtration and separation of the islets on Ficoll gradients. A remarkably high yield of purified islets has been obtained from the pig pancreas with this procedure. The islets are morphologically intact and respond to acute stimulation with glucose in Vitro.

Evidence for a sequestration of function within the area postrema based on scanning electron microscopy and the penetration of horseradish peroxidase.

SummaryScanning electron microscopy and the penetration of horseradish peroxidase, especially from the ventricular surface, has been utilized to determine the distinctive properties of the posterior portion of the area postrema. This part of the organ is characterized by a non-ciliated surface composed of flattened cells, which appear less permeable to cisternally injected peroxidase than the ciliated ependymal cells covering the anterior part of the area postrema. However, more diffuse and rapid penetration of peroxidase into the posterior region is achieved by way of the perivascular spaces which appear in direct communication with the CSF. No such filling is noted in the anterior area postrema. The posterior portion also contains cells which appear to be rapidly penetrated by horseradish peroxidase and which may be important as a sensing mechanism. The chief distinction of the anterior part of the area postrema appears to be the presence of vascular connections with the choroid plexus.

An improved method for the isolation of islets from the beef pancreas.

An improved method for the isolation of islets from the beef and pig pancreas Is described. The procedure involves the use of strips of Velcro that retain the partially-digested collagen during the isolation of islets by the collagenase technique. The spiny portion of the Velcro Is ideally suited to retain the collagen and yet permit the separation of islets from the pancreatic parenchyma. A remarkably high yield of islets has been obtained from the beef and pig pancreas USing this procedure.

A new site for islet transplantation ― A peritoneal-omental pouch

A new site for islet transplantation is described. A peritoneal-omental pouch was constructed in diabetic rats by encasing the omentum in a pouch formed from a strip of parietal peritoneum obtained from the recipient. Isografts of rat islets placed in the pouch maintained normoglycemia in the recipients and removal of the pouch resulted in a rapid return to a diabetic state. This site may be applicable to the transplantation of islets in human diabetes.

Pulsatile Insulin Secretion from Isolated Human Pancreatic Islets

Insulin secretion from the pancreas is pulsatile. The precise site and function of the pacemaker that regulates insulin periodicity in humans have not been determined. We isolated human pancreatic islets from five cadaver organ donors by collagenase digestion and density gradient purification. After 24 h of culture in CMRL-1066 medium at 37°C, aliquots of 200 islets were perifused (1 ml/min for 120 min) with glucose and other secretagogues in oxygenated Krebs-Ringer bicarbonate solution at 37°C. Samples for insulin measurement were taken every minute, and insulin secretion was analyzed by the Clifton and Steiner cycle detection technique. With 3.3 mM glucose (n = 17), insulin oscillations were demonstrated with a periodicity of 9.8 ± 0.1 min (means ± SE), mean amplitude was 16.8 ± 1.8 pM, and overall mean insulin release was 43.8 ± 4.2 pM. With 16.7 mM glucose (n = 14), no change of insulin periodicity was observed (10.2 ± 0.9 min), mean amplitude was 41.4 ± 10.2 pM (P < 0.01 vs. 3.3 mM glucose), and mean insulin release was 118.2 ± 19.2 pM (P < 0.01 vs. 3.3 mM glucose). Both at 3.3 and 16.7 mM glucose, the addition of 1.4 mM glucagon (n = 4), 15 mM arginine (n = 4), or 100 µg/ml tolbutamide (n = 4) caused no change of insulin periodicity but enhanced mean amplitude and mean insulin release compared with glucose alone. These results show that a pacemaker is located within the islets that regulates pulsatile insulin secretion in humans; the pacemaker is remarkably stable, because its periodicity is not affected by factors altering insulin secretion.

Long-term perfusion of isolated rats islets in vitro.

A perfusion system is described for long-term maintenance of isolated rat islets in vitro. This system permits the monitoring of the pattern, rate, and amount of insulin secretion following repeated, acute stimulations with glucose during the period of culture. Fibroblastic proliferation did not occur, thus making is possible to reclaim the islets for biochemical and morphologic studies at the conclusion of the experiments. Maintenance of the islets with a low concentration of glucose (1.0 mg./ml.) resulted in a marked decline in insulin secretion following acute stimulations with glucose (5.0 mg./ml.) during an eight-day interval. Stimulation with 10 mM theophylline and 5.0 mg./ml. glucose on day 9 resulted in enhanced insulin release. The decline in glucose sensitivity occurred even more rapidly when the islets were maintained in the presence of a lower concentration of glucose (0.5 mg./ml.). The pattern of insulin release was altered with an absence of a first phase of secretion. Adenylate cyclase activity of islets maintained with 0.5 mg./ml. glucose for four days was significantly decreased in comparison with islets from fed rats and islets maintained with 2.5 mg./ml. glucose. A means of maintaining the same biphasic pattern and amount of glucose-induced insulin release was achieved by using alternating levels of glucose (1.5 and 2.5 mg./ml) for maintenance of the islets during a 36-day interval.

EFFECT OF TRANSPLANTATION SITE AND αL3T4 TREATMENT ON SURVIVAL OF RAT, HAMSTER, AND RABBIT ISLET XENOGRAFTS IN MICE

Rat, hamster, and rabbit islets were transplanted into diabetic C57BL/B6 mice. The effect on islet xenograft survival of low-temperature culture of the donor islets, alpha L3T4 treatment of the recipients for seven days, and transplantation of the grafts either in the renal capsule or in the liver via the portal vein was determined. Renal capsule transplants of control rat, hamster, and rabbit islets cultured at 37 degrees C for one day produced normoglycemia in the recipients, with the mean survival time (MST) of the grafts ranging from 14 to 19 days. Low temperature culture alone did not produce a significant increase in the survival time. Treatment of the recipients with alpha L3T4 produced a marked prolongation of xenograft survival for all three species receiving renal subcapsular transplants of control or low temperature cultured islets. The range of MST* was from 34 to 46 days. The intrahepatic site produced an even further prolongation of the survival of concordant rat islet xenografts treated in this manner, with 60% of the recipients still normoglycemic at 100 days after transplantation. This enhancing effect of the intrahepatic site on survival did not occur with the discordant xenografts of hamster and rabbit islets.

Prolongation of islet xenograft survival by in vitro culture of rat megaislets in 95% O2

Individual rat islets could be aggregated into single megaislets in vitro and the megaislets remained morphologically and functionally intact after a 7-day period of culture in the presence of 95% O2 and 5% CO2. Cultured rat megaislets transplanted beneath the renal capsule of diabetic mice produced normoglycemia in the recipients and the survival of the xenografts was markedly prolonged by the 7-day exposure to a high oxygen tension. A single injection of antilymphocyte sera to mouse and rat lymphocytes into the recipients receiving cultured megaislets did not produce a further increase in the percentage of survival of the grafts at 70 days after transplantation. Lymphoid aggregates were present around xenografts of cultured negaislets at 60 and 90 days after transplantation. This lymphoid reaction did not interfere with the function of the xenografts since the recipients were normoglycemic and removal of the grafts resulted in a rapid return to a diabetic state. Intraportal and intrasplenic transplants of cultured rat megaislets did not survive as long as the xenografts of megaislets transplanted beneath the renal capsule. The renal subcapsule site apparently provided some immunological advantage for delaying acute rejection since transplants of individual, fresh rat islets survived for twice as long under the renal capsule as compared with intraportal transplants of fresh rat islets.

Prolonged survival of discordant porcine islet xenografts

Purified porcine islets were prepared by collagenase digestion and density gradient purification, and transplanted under the kidney capsule of C57B/B6 mice with streptozotocin-induced diabetes which were receiving varying temporary immunosuppressive therapies. Islets that had been cultured for 1 day at 37 degree C were rejected after : 9+/-0.1 (mean+/-SE) days in control mice: 14+/-3 days in mice receiving mouse antilymphocyte serum (MLS) plus porcine antilymphocyte serum (PLS) on day of transplant (day 0); 43+/-6 days in mice treated for 1 week with anti-CD4 antibody (aCD4); 36+/-4 days in mice given aCD4 for 1 week plus PLS on days 0 and 7; 47+/-3 days in mice treated with aCD4 for 1 week plus MLS and PLS on day 21. Porcine islet survival in these latter three groups was significantly (P<0.01) and similarly longer than in the control and MLS plus PLS groups. Then, we transplanted islets that had been either cultured at 24 degrees C for 7 days or cryopreserved into 7-day aCD4-treated mice, to evaluate whether low temperature culture or the freezing-thawing procedure could affect survival. Neither 7-day, low temperature culture (mean survival time: 37+/-2 days) nor cryopreservation (mean survival time: 39+/-2 days) prolonged islets function further. Thus, the present study demonstrates that prolonged survival can be achieved with discordant porcine islet xenografts, and shows the greater efficacy of aCD4 treatment, which was not improved by additional immunosuppressive therapies we tested, nor by culture or cryopreservation of the islets.