Isolation of Human Islets from Partially Pancreatectomized Patients

Abstract

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors 2 . Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.To extend the applicability of our protocol to include the occasional case in which a large amount (>15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber 3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO 2 at 37 °C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research. Video LinkThe video component of this article can be found at https://www.jove.com/video/2962/ Protocol 1. Pancreatic tissue collection in the operating room 1. The surgeon performs a partial resection of the pancreas. 2. After placing the pancreatic specimen in a box on ice, deliver it immediately to the pathologist. Tissue selection for islet isolation1. The pathologist selects tissue that appears soft and healthy, retaining the damaged tissue for diagnostic purposes. Fibrotic tissue and specimens offering less than 2 g of usable tissue are excluded from islet isolation. 2. Immerse the pancreatic tissue in Euro Collins Solution and deliver on ice to the laboratory. Isolation of human islets1. Weigh the pancreatic tissue, then place it into a 10 cm dish. 2. Put 150 ml RPMI media in a 500 ml flask. 3. In a 250 ml flask, prepare the digestive enzyme solution by combining 130 ml RPMI media with 100 mg/ml DNase and 20 ml of 5mg/ml Liberase RI. Dr...

Scientific Advances in Diabetes: The Impact of the Innovative Medicines Initiative

“…Additionally, several novel tools were identified for diabetes, T1D, T2D, diabetic complications, and genetic research. The tools for diabetes intended to diagnose and monitoring disease progression [IMIDIA ( 44 – 49 ), DIRECT ( 50 ), and RHAPSODY ( 51 )]. The tools designed for T1D were focused on monitor β-cell function, screen individuals at high risk, and select the more benefic intervention [INNODIA ( 15 , 52 , 53 )].…”

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Human Islet Isolation and Distribution Efforts for Clinical and Basic Research

“…Human islets are typically isolated from the pancreas of a cadaveric donor, although they can also be derived from live donors that have undergone partial pancreatectomy [9][10][11]. The isolation of islets from the human pancreas is a difficult and sensitive process that requires a specialized and licensed facility, as well as the combined efforts of multiple trained personnel.…”

Metabolically phenotyped pancreatectomized patients as living donors for the study of islets in health and diabetes

“…This method greatly reduced enzymatic and mechanical damage done to the islets during the isolation procedure, but also facilitated an otherwise very labour-intensive process. It also reduced the amount of necessary tissue input to obtain a meaningful number of human islets, which also enabled their collection from organs derived from partial pancreatectomies [7]. Despite the various improvements to the protocol introduced by numerous researchers throughout the following decades, purity of the islet preparation remains one of the significant setbacks of this method of obtaining islets for diabetes research.…”