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Ex vivo maturation of islet cells

A mature technology of islet cells, applied in the field of ex vivo maturation of islet cells, can solve the problems of costing up to two to three months, unsatisfactory, variable glucose responsiveness, etc.

Inactive Publication Date: 2014-08-27
ISLET SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these immature islets fail to secrete glucose-responsive insulin for at least two weeks after their isolation and may take up to two to three months to become glucose-responsive after transplantation into the host (Korsgran O, et al. (1988) Transplantation 1988 ;45:509-14)
Therefore, islets from neonatal and fetal pancreas are clinically unsatisfactory due to their variable glucose responsiveness and the long time it takes to mature

Method used

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  • Ex vivo maturation of islet cells
  • Ex vivo maturation of islet cells
  • Ex vivo maturation of islet cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. Islet removal and isolation

[0047] Weaning piglets were used as a donor source of immature islets. A typical islet preparation method uses ten Yorkshire piglets aged 14 to 26 days. Pancreas were removed from piglets by quick surgical harvest (ie, less than five minutes) and placed in ice-cold organ preservation solution (Corning Cellgro). The ischemic time throughout the cooling was limited to 20 minutes. Harvested pancreases were pooled and dissected as follows. After freezing, animal fat and lymphatic tissue around the pancreas are excised, followed by finely mincing until the diameter of the tissue section is usually 0.5 mm to 1.0 mm. Continue the cutting process using standard scissors and agitation techniques to mince the pancreatic tissue. Minced tissue was added to an enzyme mix of low-dose Clzyme collagenase MA / BP protease (75-250 mg / pancreas, VitaCyte LLC, Indianapolis, IN) and allowed to digest at 37°C with gentle shaking and rotation (40-250 ...

Embodiment 2

[0048] Example 2. Recovery of isolated islets

[0049] For 48 hours after the first isolation, islet clusters were cultured in a new tissue culture medium called recovery maturation medium. The first step in preparing recovery maturation medium involves obtaining Ham's F-12 / Medium199 basal medium (F-12 / 199 medium) in which Ham's F-12 and Medium199 components are present in a 1:1 ratio. In these studies, Ham's F-12 and Medium199 powders (both purchased from Cellgro, Inc.) were added at a weight / volume (w / v) concentration of 0.815% each (ie, 8.15 g in 1 L of water) : 2000 and cGMP guidelines (Thermo Scientific Inc., Waltham, MA) sterile water to prepare F-12 / 199 medium. Add to this F-12 / 199 medium afterwards: 5.5% (w / v) porcine serum (Lampire Biological Laboratories, Inc., Pipersville, PA); 2.5 * 10 -3 % (w / v) gentamicin sulfate (Fisher Bioreagents, Thermo Fisher Scientific, Inc., Waltham, MA); 0.062% (w / v) glutathione tripeptide (Acros Organics, Thermo Fisher Scientific, Inc....

Embodiment 3

[0050] Example 3. Maturation of resected islets

[0051] After 48 hours of culture in Recovery Maturation Medium, islets were removed from Recovery Maturation Medium, centrifuged at 200 xg for two minutes, and then resuspended in a modification of Recovery Maturation Medium referred to herein simply as "Maturation Medium". Specifically, the difference between the recovery mature medium and the mature medium is that the mature medium has half the amount of Pefabloc TM and DNase alpha. Islets were cultured in maturation medium until the islets were fully mature, a period of about eight to nine days, during which time 50% of the medium was changed every 48 hours. Full maturation of islets was defined according to histochemical and functional assays described in the Examples below. When the islets reach full maturity, they can be cultured long-term in "supplemented maturation medium", which is a maturation medium that contains an additional 5 mM concentration of calcium. Additi...

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Abstract

The invention relates to methods for promoting maturation of islet cells from pre-weaned mammals for the purpose of optimizing the islets for their use as donor tissue for xenotransplantation. The method of the invention removes the pancreas from donor animals and reduces the pancreas tissue to fragments that are greater than the size of an intact islet while retaining islets in their whole, insulin-producing condition. The method of the invention also serially cultures the digested tissue in novel maturation media that enhance the glucose responsiveness of the cultured islets, and selects islets that are sufficiently glucose-responsive for use in transplantation procedures.

Description

[0001] Cross References to Related Applications [0002] Priority is claimed to US Application Serial No. 61 / 540,288 and US Application Serial No. 61 / 540,293, filed September 28, 2011, the entire disclosures of which are incorporated herein by reference. technical field [0003] The present invention relates to methods for isolating and culturing immature Islets of Langerhans to maturity. Background technique [0004] Diabetes is a group of disorders with several common features, of which increased blood glucose is the most obvious. The four most common types of diabetes are type 1 diabetes (T1D), type 2 diabetes, secondary diabetes and gestational diabetes. T1D is an insulin deficiency disorder with onset predominantly in infancy and if left untreated is characterized by hyperglycemia. T2D is a disorder in which insulin insensitivity is combined with the inability of pancreatic insulin secretion to compensate for insulin insensitivity. Secondary diabetes occurs after dam...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/567
CPCC12N2500/25C12N2500/44C12N5/0676C12N2500/38C12N2501/91C12N2501/734C12N2501/73C12N2501/335C12N2501/33A61P3/10
Inventor 乔纳森·RT·莱基
Owner ISLET SCI
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