Cryopreservation of Human Pancreatic Islets

Lakey, Jonathan R. T.; Burridge, Philip W.; Rajotte, Ray V.

doi:10.1177/1522162802005005001

Cited by 11 publications

(16 citation statements)

References 80 publications

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“…Similarly, by selecting for largest diameter, smooth-surfaced single cells after several days in culture, it is likely that we were choosing for study the most viable cells and avoiding the bulk of cells undergoing apoptosis. Recent improvements in cryopreservation technique include: reduction in the concentration of DMSO as cryopreservant [29], substitution of ethylene glycol or trehalose as the cryopreservant [30,31], and addition of antioxidants such as beraprost sodium, butylated hydroxyanisole or ascorbic acid-2 glucoside to the cryopreservant [32][33][34].…”

Section: Discussionmentioning

confidence: 99%

Maintenance of stimulus-secretion coupling and single beta-cell function in cryopreserved-thawed human islets of Langerhans

Misler

Dickey

Barnett

2005

Pflugers Arch - Eur J Physiol

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Studies of stimulus-secretion coupling in human beta-cells have been hampered by poor availability of tissue due to variability of the supply of cadaver pancreati and in the adequacy of enzymatic liberation of islets as well as by the shunting of isolates into transplant trials. Here we establish that aliquots of islets, several from high-quality but low-yield islet isolates (50,000-100,000 islets), cryopreserved and then thawed as needed, respond to glucose in a calcium- and metabolic-dependent fashion. Insulin secretion is modulated by blockers of voltage-dependent Na+ and Ca2+ channels, and paracrine hormones (glucagon and somatostatin) in manners indistinguishable from fresh tissue preparations. Using single-cell electrophysiological and electrochemical assays we demonstrate that single beta-cells from cryopreserved islets display (1) stimulus-depolarization coupling based on rapid closure of K+ (ATP) channels; (2) action potential electrogenesis with upstrokes based on voltage-dependent Na and Ca currents; and (3) Ca2+ entry-mediated depolarization-exocytosis coupling sustained over multiple bouts of stimulation and modulated by paracrine hormones. All of these features are indistinguishable from those seen in single cells from freshly harvested islets. These results support the utility of cryopreservation, even of low-yield but functional isolates, as a means of ensuring a steady source of repeatedly accessible tissue for research on normal and diabetic islets.

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Section: Discussionmentioning

confidence: 99%

Maintenance of stimulus-secretion coupling and single beta-cell function in cryopreserved-thawed human islets of Langerhans

Misler

Dickey

Barnett

2005

Pflugers Arch - Eur J Physiol

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“…Cell viabilities after cryopreservation were as‐sessed by an AO/PI assay, as previously reported (10). Thawed human islets after cryopreservation were suspended with CRMI medium supplemented with 10% FBS and seeded on six‐well plates at a density of 100 islets/well.…”

Section: Methodsmentioning

confidence: 99%

Maintenance of Glucose‐sensitive Insulin Secretion of Cryopreserved Human Islets with University of Wisconsin Solution and Ascorbic Acid‐2 Glucoside

et al. 2004

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Normal human islet cells are an ideal source for pancreas-targeted cell therapies, but the availability of human donor pancreata for islet isolation is severely limited. To effectively utilize such scarce donor organs for cell therapies, it is crucial to develop an excellent isolation, effective cryopreservation, and efficient gene transfer techniques for the transportation of isolated cells. In the present study, we investigate the effect of University of Wisconsin (UW) solution and ascorbic acid-2 glucoside (AA2G) on the cryopreservation of human islets. We also evaluate the gene transfer efficiency of a lentiviral vector expressing the E. coli LacZ gene, Lt-NLS/LacZ, in human islets. Human islets were isolated with a standard digestion method at the University of Alberta. Isolated islets were transported to Japan for 40 h and then subjected to cryopreservation experiments. The following preservation solutions were tested: UW solution with 100 micro g/mL of AA2G, UW solution, 100% fetal bovine serum (FBS), and CMRL supplemented with 10% FBS. Following three months of cryopreservation, the islets were thawed and analyzed for viability, glucose-sensitive insulin secretion, proinsulin gene expression profile, and in vivo engraftment. The islets were also subjected to monolayer formation with 804G-cell-line-derived extracellular matrix (ECM), followed by Lt-NLS/LacZ transduction. The viability, morphology, glucose-sensitive insulin secretion, proinsulin gene expression, and monolayer formation efficiency of the thawed cryopreserved islets are significantly better maintained by the use of UW solution. When AA2G (100 microg/mL) is combined with UW, such parameters are further improved. The adequate engraftment of UW + AA2G-cryopreserved human islets is achieved in the liver of nude mice. Efficient Lt-NLS/LacZ transduction is identified in monolayered islets cryopreserved with UW solution with AA2G. The present work demonstrates that the combination of UW solution with AA2G (100 microg/mL) would be a useful cryopreservation means for human islets. Human islets monolayer-cultured with 804G-derived ECM are efficiently transduced with a lentiviral vector Lt-NLS/LacZ.

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“…[ 19 ] These results demonstrated that suboptimal cryopreservation process can cause cryoinjuries (i.e., ice injury and solute injury) to the cells and loss of islet functionalities, which warranted the need of further improvements of the islet cryopreservation protocols. [ 3,4,45 ] To understand how cryopreservation and microencapsulation affect the islet functionality, we fi rst measured the ATP and static insulin release values of bare islets (B), encapsulated islets (E), bare islets cryopreserved and stored at −80 °C for 1 or 7 d (B1d or B7d), encapsulated islets cryopreserved and stored at −80 °C for 1 or 7 d (E1d or E7d), and encapsulated islets cryopreserved with trehalose (additional CPA to the routinely used DMSO medium) and stored at −80 °C for 1 or 7 d (ET1d or ET7d) for comparison studies. Figure 3 shows the ATP value of islets under different protocols for encapsulated/bare islets with or without cryopreservation.…”

Section: Atp and Static Insulin Release Measurementsmentioning

confidence: 99%

“…[ 1 ] In the last decade, transplantation of pancreatic islets, such as the well-known "Edmonton protocol", has been considered as the most physiologically advantageous solution for glucose homeostasis in clinical treatments of selected patients with type 1 diabetes mellitus, which can eliminate insulin injection, reverse neurovascular complications, and even prevent end-stage organ failure. [2][3][4][5][6] In such treatments, suffi cient islet equivalents with high quality are critical for positive clinical outcomes. For example, the Edmonton protocol requires a cumulative islet mass of 10 000 islet equivalents or more per kilogram of body weight of the recipient.…”

Section: Introductionmentioning

confidence: 99%

Sensing and Sensibility: Single‐Islet‐based Quality Control Assay of Cryopreserved Pancreatic Islets with Functionalized Hydrogel Microcapsules

Chen

Shu

Gao

et al. 2015

Adv Healthcare Materials

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Despite decades of research and clinical studies of islet transplantations, fi nding simple yet reliable islet quality assays that correlate accurately with in vivo potency is still a major challenge, especially for real-time and singleislet-based quality assessment. Herein, proof-of-concept studies of a cryopreserved microcapsule-based quality control assays are presented for single islets. Individual rat pancreatic islets and fl uorescent oxygen-sensitive dye (FOSD) are encapsulated in alginate hydrogel microcapsules via a microfl uidic device. To test the susceptibility of the microcapsules and the FOSD to cryopreservation, the islet microcapsules containing FOSD are cryopreserved and the islet functionalities (adenosine triphosphate, static insulin release measurement, and oxygen consumption rate) are assessed after freezing and thawing steps. The cryopreserved islet capsules with FOSD remain functional after encapsulation and freezing/thawing procedures, validating a simple yet reliable individual-islet-based quality control method for the entire islet processing procedure prior to transplantation. This work also demonstrates that the functionality of cryopreserved islets can be improved by introducing trehalose into the routinely used cryoprotectant dimethyl sulfoxide. The functionalized alginate hydrogel microcapsules with embedded FOSD and optimized cryopreservation protocol presented in this work serve as a versatile islet quality assay and offer tremendous promise for tackling existing challenges in islet transplantation procedures.

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Cryopreservation of Human Pancreatic Islets

Cited by 11 publications

References 80 publications

Maintenance of stimulus-secretion coupling and single beta-cell function in cryopreserved-thawed human islets of Langerhans

Maintenance of stimulus-secretion coupling and single beta-cell function in cryopreserved-thawed human islets of Langerhans

Maintenance of Glucose‐sensitive Insulin Secretion of Cryopreserved Human Islets with University of Wisconsin Solution and Ascorbic Acid‐2 Glucoside

Sensing and Sensibility: Single‐Islet‐based Quality Control Assay of Cryopreserved Pancreatic Islets with Functionalized Hydrogel Microcapsules

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