Automated Large-Scale Isolation, in Vitro Function and Xenotransplantation of Porcine Islets of Langerhans

Marchetti, Piero; Finke, Edward H.; Gerasimidi-Vazeou, Andriani; Falqui, Luca; Scharp, David W.; Lacy, Paul E.

doi:10.1097/00007890-199108000-00005

Cited by 62 publications

(21 citation statements)

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“…Total islet equivalents (IE) isolated varied between 950 and 3724 IE/g. Our results were similar to those of other reports (1642-3551 IE/g pancreas) (Marchetti et al 1991;Ricordi et al 1986) in these specific isolations. Nevertheless, this number of IE may be skewed by individual counting error or by very large islets.…”

Section: Lmsupporting

confidence: 93%

“…To optimize mass purification in the COBE, a Euroficoll test gradient was performed for each manipulation (Kerr-Conte et al 1994). Purified islets were cultivated in RPMI 1640, 1 g/l glucose with 10% fetal calf serum, 10 mM niacinamide, and antibiotics at 37°C in equilibrium with 5% CO 2 (Marchetti et al 1991). Triplicate samples were taken from the final digestate in order to quantitatively assess the islets.…”

Section: Methodsmentioning

confidence: 99%

“…To date, the pig pancreas, due to its anatomical and physiological similarities with human pancreas, seems the organ of choice (Marchetti et al 1991;Ricordi et al 1986). Assessment of islets isolated from the pancreas of large mammals includes quantitative and qualitative criteria, among which are islet number and volume, purity, morphological integrity, and test of islet viability.…”

Section: Introductionmentioning

confidence: 99%

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Immunohistochemical and ultrastructural study of adult porcine endocrine pancreas during the different steps of islet isolation

Vantyghem¹,

Kerr–Conte²,

Pattou³

et al. 1996

Histochem Cell Biol

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Treatment of diabetes mellitus by transplantation of isolated pancreatic islets could constitute an alternative to human pancreas allograft. Before transplantation, porcine islets are submitted to a procedure of isolation and purification. The quality of islets through these different steps may be assessed by morphological and functional studies. The aim of this work was the histological characterization of the four main cell types of porcine adult endocrine islets during the different steps of the isolation procedure using immunohistochemistry (IHC) applied in light (LM) and electron microscopy (EM). In fresh pancreas, islets were various sizes and shapes in LM. The number was not found different between the different portions of the pancreas. In IHC, insulin (Ins)-secreting cells accounted for the majority of the islet cells, while glucagon(Glu)-somatostatin (Som)- and polypeptide(PP)-immunoreactive cells, in decreasing number, were found in the mantle around the core of Ins-cells. In EM, B-cells contained poly-hedric granules with a dense central core and clear halo. Glu granules were spherical and very dense. D-cells and PP-cells were characterized by numerous granules, rather spherical and of inequal density for Som and more ellipsoidal for PP granules. After purification in Euroficoll, in EM, the four cellular types remained recognizable, but underwent vacuolization, mitochondrial swelling, and enlargement of intercellular spaces. After 3 days of culture on plastic dishes, as on Biopore membranes in a Millicell insert, microvilli appeared and vacuolization increased in EM. At the seventh day of culture, in EM, most of the cells were lysed in contrast to LM where at the same time, the four cell types were clearly identified by IHC but only in collagen matrix. Important discrepancies were noticed between LM and EM. This fact emphasizes the complementarity of morphological and functional studies in assessment of the quality of an islet isolation.

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Section: Lmsupporting

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Immunohistochemical and ultrastructural study of adult porcine endocrine pancreas during the different steps of islet isolation

Vantyghem¹,

Kerr–Conte²,

Pattou³

et al. 1996

Histochem Cell Biol

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“…This occurrence may be related to the necessity of improved insulin secretory response to glucose stimulation by cAMP-raising agent (eg, forskolin) used in other laboratories for freshly isolated pig islets. 16,17 However, the poor insulin-secreting capacity of isolated pig islets in vitro does not necessarily imply a lack of function in vivo. 18 The optimal condition for islet cryopreservation in freezing and thawing periods are still under investigation.…”

Section: Discussionmentioning

confidence: 99%

Untitled

Kinasiewicz

Antosiak-Iwańska²,

Godlewska³

et al. 2018

Exp Clin Transplant

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Objectives: Immunoisolation of pancreatic islets of Langerhans performed by the encapsulation process may be a method to avoid immunosuppressive therapy after transplant. The main problem related to islet transplant is shortage of human pancreata. Resolution of this obstacle may be cryopreservation of encapsulated islets, which enables collection of sufficient numbers of isolated islets required for transplant and long-term storage. Here, we assessed the ability of encapsulated islets to function after longterm banking at low temperature. Materials and Methods: Islets of Langerhans isolated from rat, pig, and human pancreata were encapsulated within alginate-poly-L-lysine-alginate microcapsules. Cryopreservation was carried out using a controlled method of freezing (Kriomedpol freezer; Kriomedpol, Warsaw, Poland), and samples were stored in liquid nitrogen. After 10 years, the samples were thawed with the rapid method (with 0.75 M of sucrose) and then cultured. Results: We observed that microcapsules containing islets maintained their shape and integrity after thawing. During culture, free islets were defragmented into single cells, whereas encapsulated islets were still round in shape and compact. After 1, 4, and 7 days of culture of encapsulated islets, the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tests showed increased mitochondrial activity. After they were thawed, the insulin secretion capacity was comparable with that obtained with fresh islets.Conclusions: Cryopreservation and storage of free and microencapsulated islets were possible for about 10 years, although only encapsulated islets retained viability and secretory properties.

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“…Isolation of rat pancreas islets have been first described by Lacy and Kostianovsky (1967) and after that many other procedures have been reported for pancreatic islet isolation based on this technique (Ricordi et al 1988;Wolters et al 1990;Marchetti et al 1991). However there is a low degree of reproducibility for described techniques besides isolation of significant number of pancreatic islets is not easy due to various factors involved in the process.…”

Section: Introductionmentioning

confidence: 99%

Optimizing Conditions for Rat Pancreatic Islets Isolation

2005

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Many procedures have been described for rat pancreatic islet isolation. Several factors contribute to the pancreatic islet isolation outcome. One of the main problems in islet isolation procedure is the formation of a viscouse, gellike structure during collagenase digestion which entraps the free islets and decrease islet yield after density gradient purification. This issue has not been addressed in most techniques described for rat islet isolation. We examined effect of various factors to eliminate formation of gellike material and improve the islets yields. Islet isolation was performed on 26 adult male Wistar Albino rats weighing between 280 and 350 g. We have observed that several factors affect pancreatic islet isolation. Optimum Collagenase enzyme concentration, maintaining pH range between 7.7 and 7.9 in digestion solution, incubation temperature at 38+/-1 degrees C and addition of Calcium ion decreased the formation of gellike materials and increased islet yield. Addition of Glycerol as a gelatin solvent has also been helpful in the reduction or complete elimination of gellike material. Precise optimization of rat islet isolation procedure is useful to improve the islet yield in islet transplantation studies.

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Automated Large-Scale Isolation, in Vitro Function and Xenotransplantation of Porcine Islets of Langerhans

Cited by 62 publications

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Immunohistochemical and ultrastructural study of adult porcine endocrine pancreas during the different steps of islet isolation

Immunohistochemical and ultrastructural study of adult porcine endocrine pancreas during the different steps of islet isolation

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Optimizing Conditions for Rat Pancreatic Islets Isolation

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