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Isolation of Human Islets from Partially Pancreatectomized Patients
- Source :
- Journal of Visualized Experiments : JoVE
- Publication Year :
- 2011
- Publisher :
- MyJove Corporation, 2011.
-
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.
- Subjects :
- medicine.medical_specialty
Pathology
medicine.medical_treatment
General Chemical Engineering
Cytological Techniques
Ficoll
030209 endocrinology & metabolism
human islets
General Biochemistry, Genetics and Molecular Biology
Pathogenesis
03 medical and health sciences
Islets of Langerhans
0302 clinical medicine
Pancreatectomy
Diabetes mellitus
partial pancreatectomy
Internal medicine
medicine
Humans
Pancreas
Issue 53
030304 developmental biology
0303 health sciences
geography
geography.geographical_feature_category
Common bile duct
General Immunology and Microbiology
business.industry
General Neuroscience
medicine.disease
Islet
3. Good health
medicine.anatomical_structure
Endocrinology
Diabetes Mellitus, Type 2
Pancreatitis
Medicine
Digestion
business
human islet isolation
Subjects
Details
- ISSN :
- 1940087X
- Database :
- OpenAIRE
- Journal :
- Journal of Visualized Experiments
- Accession number :
- edsair.doi.dedup.....1bb72e95312ca628ebb0c822246c340d
- Full Text :
- https://doi.org/10.3791/2962-v