scholarly journals Comparison of Purification Solutions with Different Osmolality for Porcine Islet Purification

Cell Medicine ◽  
2017 ◽  
Vol 9 (1-2) ◽  
pp. 53-59 ◽  
Author(s):  
Chika Miyagi-Shiohira ◽  
Naoya Kobayashi ◽  
Issei Saitoh ◽  
Masami Watanabe ◽  
Yasufumi Noguchi ◽  
...  
Keyword(s):  
Salt Solution ◽  
Stimulation Index ◽  
Human Islets ◽  
Human Islet ◽  
Low Density ◽  
Islet Isolation ◽  
Continuous Density ◽  
Yield Rate ◽  
Purification Efficiency ◽  
Porcine Islet

The osmolality of the purification solution is one of the most critical variables in human islet purification during islet isolation. We previously reported the effectiveness of a combined continuous density/osmolality gradient for the supplemental purification of human islets. We herein applied a combined continuous density/osmolality gradient for regular purification. The islets were purified with a continuous density gradient without osmolality preparation [continuous density/normal osmolality (CD/NO)] or continuous density/osmolality solution with osmolality preparation by 10× Hank's balanced salt solution (HBSS) [continuous density/continuous osmolality (CD/CO)]. The osmolality of the low-density solution was 400 mOsm/kg in both groups and that of the high-density solution was 410 mOsm/kg in the CD/NO group and 500 mOsm/kg in the CD/CO group. Unexpectedly, we noted no significant differences between the two solutions in terms of the islet yield, rate of viability and purity, score, stimulation index, or the attainability and suitability of posttransplantation normoglycemia. Despite reports that the endocrine and exocrine tissues of pancreata have distinct osmotic sensitivities and that high-osmolality solutions result in greater purification efficiency, the isolation and transplant outcomes did not markedly differ between the two purification solutions with different osmolalities in this study.

scholarly journals Comparison of Clostripain and Neutral Protease as Supplementary Enzymes for Human Islet Isolation

2018 ◽  
Vol 28 (2) ◽  
pp. 176-184 ◽  
Author(s):  
Heide Brandhorst ◽  
Paul R. Johnson ◽  
Johanna Mönch ◽  
Manfred Kurfürst ◽  
Olle Korsgren ◽  
...  
Keyword(s):  
Islet Transplantation ◽  
Stimulation Index ◽  
Effective Means ◽  
Insulin Response ◽  
Human Islets ◽  
Human Islet ◽  
Neutral Protease ◽  
Islet Isolation ◽  
Clinical Islet Transplantation ◽  
Glucose Challenge

Although human islet transplantation has been established as valid and safe treatment for patients with type 1 diabetes, the utilization rates of human pancreases for clinical islet transplantation are still limited and substantially determined by the quality and composition of collagenase blends. While function and integrity of collagenase has been extensively investigated, information is still lacking about the most suitable supplementary neutral proteases. The present study compared islet isolation outcome after pancreas digestion by means of collagenase used alone or supplemented with either neutral protease (NP), clostripain (CP), or both proteases. Decent amounts of islet equivalents (IEQ) were isolated using collagenase alone (3090 ± 550 IEQ/g), or in combination with NP (2340 ± 450 IEQ/g) or CP (2740 ± 280 IEQ/g). Nevertheless, the proportion of undigested tissue was higher after using collagenase alone (21.1 ± 1.1%, P < 0.05) compared with addition of NP (13.3 ± 2.2%) or CP plus NP (13.7 ± 2.6%). Likewise, the percentage of embedded islets was highest using collagenase only (13 ± 2%) and lowest adding NP plus CP (4 ± 1%, P < 0.01). The latter combination resulted in lowest post-culture overall survival (42.7 ± 3.9%), while highest survival was observed after supplementation with CP (74.5 ± 4.8%, P < 0.01). An insulin response toward glucose challenge was present in all experimental groups, but the stimulation index was significantly decreased using collagenase plus NP (2.0 ± 0.12) compared with supplementation with CP (3.16 ± 0.4, P < 0.001). This study demonstrates for the first time that it is possible to isolate significant numbers of human islets combining collagenase only with CP. The supplementation with CP is an effective means to substantially reduce NP activity, which significantly decreases survival and viability after culture. This will facilitate the manufacturing of enzyme blends with less harmful characteristics.

scholarly journals Evaluation of Islet Purification Methods for Making a Continuous Density Gradient and Loading Tissue

Cell Medicine ◽  
2018 ◽  
Vol 10 ◽  
pp. 215517901773309 ◽  
Author(s):  
Nana Ebi ◽  
Chika Miyagi-Shiohira ◽  
Eri Hamada ◽  
Yoshihito Tamaki ◽  
Mariko Masamoto ◽  
...  
Keyword(s):  
Density Gradient ◽  
Stimulation Index ◽  
High Yield ◽  
Islet Isolation ◽  
Pancreatic Islet Transplantation ◽  
Porcine Pancreas ◽  
Purification Method ◽  
Continuous Density ◽  
Yield Rate ◽  
Purification Methods

Islet purification is one of the most important steps of islet isolation for pancreatic islet transplantation. We previously reported that a purification method using large plastic bottles effectively achieved a high yield of islets from porcine pancreas. In this study, we evaluated the methods for making a continuous density gradient and loading tissue. One method involved loading digested tissue on top of a continuous density gradient (top loading). The other method involved mixing digested tissue with low-density solution and then making a continuous gradient (mixed loading). There were no significant differences between the 2 purification methods in terms of the islet yield, rate of viability or purity, score, or in the stimulation index after purification. Furthermore, there were no marked differences in the attainability or suitability of posttransplantation normoglycemia. Our study shows the equivalency of these 2 methods of islet purification.

scholarly journals Optimal Time to Ship Human Islets Post Tissue Culture to Maximize Islet

2020 ◽  
Vol 29 ◽  
pp. 096368972097458
Author(s):  
Barbara J. Olack ◽  
Michael Alexander ◽  
Carol J. Swanson ◽  
Julie Kilburn ◽  
Nicole Corrales ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Stimulation Index ◽  
Human Islets ◽  
Human Islet ◽  
Optimal Time ◽  
Diabetes Research ◽  
Islet Function ◽  
Islet Isolation ◽  
Culture Time

Access to functional high-quality pancreatic human islets is critical to advance diabetes research. The Integrated Islet Distribution Program (IIDP), a major source for human islet distribution for over 15 years, conducted a study to evaluate the most advantageous times to ship islets postisolation to maximize islet recovery. For the evaluation, three experienced IIDP Islet Isolation Centers each provided samples from five human islet isolations, shipping 10,000 islet equivalents (IEQ) at four different time periods postislet isolation (no 37°C culture and shipped within 0 to 18 hours; or held in 37°C culture for 18 to 42, 48 to 96, or 144 to 192 hours). A central evaluation center compared samples for islet quantity, quality, and viability for each experimental condition preshipment and postshipment, as well as post 37°C culture 18 to 24 hours after shipment receipt. Additional evaluations included measures of functional potency by static glucose-stimulated insulin release (GSIR), represented as a stimulation index. Comparing the results of the four preshipment holding periods, the greatest IEQ loss postshipment occurred with the shortest preshipment times. Similar patterns emerged when comparing preshipment to postculture losses. In vitro islet function (GSIR) was not adversely impacted by increased tissue culture time. These data indicate that allowing time for islet recovery postisolation, prior to shipping, yields less islet loss during shipment without decreasing islet function.

scholarly journals Comparison of Tissue Loading Before and After the Creation of a Continuous Density Gradient in Porcine Islet Purification

Cell Medicine ◽  
2018 ◽  
Vol 10 ◽  
pp. 215517901878134 ◽  
Author(s):  
Chika Miyagi-Shiohira ◽  
Yoshiki Nakashima ◽  
Nana Ebi ◽  
Eri Hamada ◽  
Yoshihito Tamaki ◽  
...  
Keyword(s):  
Density Gradient ◽  
Stimulation Index ◽  
High Yield ◽  
Pancreatic Islet Transplantation ◽  
Post Transplantation ◽  
Porcine Pancreas ◽  
Purification Method ◽  
Continuous Density ◽  
Porcine Islet ◽  
The Impact

The purification step is one of the most important and difficult procedures in islet isolation for pancreatic islet transplantation. We previously reported that a purification method using large plastic bottles effectively achieved a high yield of islets from the porcine pancreas. In this study, we evaluated the impact of the timing of tissue loading on porcine islet purification using large plastic bottles. One method involved loading digested tissue after creating a continuous density gradient (tissue after gradient [TAG]). The other method involved loading digested tissue before creating a continuous density gradient (tissue before gradient [TBG]). There were no significant differences between TAG and TBG in terms of the islet yield, rates of viability and purity, score, and in the stimulation index after purification. Furthermore, there were no marked differences in the attainability or suitability of post-transplantation normoglycemia. Our study shows the equivalency of these two methods of islet purification.

scholarly journals Research-Focused Isolation of Human Islets From Donors With and Without Diabetes at the Alberta Diabetes Institute IsletCore

Endocrinology ◽  
2015 ◽  
Vol 157 (2) ◽  
pp. 560-569 ◽  
Author(s):  
James Lyon ◽  
Jocelyn E. Manning Fox ◽  
Aliya F. Spigelman ◽  
Ryekjang Kim ◽  
Nancy Smith ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Glycated Hemoglobin ◽  
Organ Procurement ◽  
Human Islets ◽  
Human Islet ◽  
Islet Function ◽  
Small Contribution ◽  
Islet Isolation ◽  
Diabetes Status

Abstract Recent years have seen an increased focus on human islet biology, and exciting findings in the stem cell and genomic arenas highlight the need to define the key features of mature human islets and β-cells. Donor and organ procurement parameters impact human islet yield, although for research purposes islet yield may be secondary in importance to islet function. We examined the feasibility of a research-only human islet isolation, distribution, and biobanking program and whether key criteria such as cold ischemia time (CIT) and metabolic status may be relaxed and still allow successful research-focused isolations, including from donors with type 1 diabetes and type 2 diabetes. Through 142 isolations over approximately 5 years, we confirm that CIT and glycated hemoglobin each have a weak negative impacts on isolation purity and yield, and extending CIT beyond the typical clinical isolation cutoff of 12 hours (to ≥ 18 h) had only a modest impact on islet function. Age and glycated hemoglobin/type 2 diabetes status negatively impacted secretory function; however, these and other biological (sex, body mass index) and procurement/isolation variables (CIT, time in culture) appear to make only a small contribution to the heterogeneity of human islet function. This work demonstrates the feasibility of extending acceptable CIT for research-focused human islet isolation and highlights the biological variation in function of human islets from donors with and without diabetes.

scholarly journals The Evaluation of Islet Purification Methods That Use Large Bottles to Create a Continuous Density Gradient

Cell Medicine ◽  
2017 ◽  
Vol 9 (1-2) ◽  
pp. 45-51 ◽  
Author(s):  
Chika Miyagi-Shiohira ◽  
Naoya Kobayashi ◽  
Issei Saitoh ◽  
Masami Watanabe ◽  
Yasufumi Noguchi ◽  
...  
Keyword(s):  
Stainless Steel ◽  
Density Gradient ◽  
Stimulation Index ◽  
High Density ◽  
Steel Pipe ◽  
High Yield ◽  
Low Density ◽  
Pancreatic Islet Transplantation ◽  
Continuous Density ◽  
Stainless Steel Pipe

Islet purification is one of the most important steps of islet isolation for pancreatic islet transplantation. The most common method of islet purification is density gradient centrifugation using a COBE 2991 cell processor. However, this method can damage islets mechanically through its high shearing force. We recently reported that a new purification method using large plastic bottles effectively achieves a high yield of islets from the porcine pancreas. In the present study, we evaluated the methods of making a continuous density gradient. The gradient was produced with a gradient maker and two types of candy cane-shaped stainless steel pipes. One method was to use a “bent-tipped” stainless steel pipe and to load from a high-density solution to a low-density solution, uploading the stainless steel pipe. The other method was to use a regular stainless steel pipe and to load from a low-density solution to a high-density solution, leaving the stainless steel pipe in place. There were no significant differences between the two solutions in terms of the islet yield, rate of viability or purity, score, or the stimulation index after purification. Furthermore, there were no differences in the attainability or suitability of posttransplantation normoglycemia. Our study shows the equivalency of these two methods of islet purification.

scholarly journals A Comparison of Pancreatic Islet Purification using Iodixanol with University of Wisconsin Solution and with Na-Lactobionate and Histidine Solution

Cell Medicine ◽  
2018 ◽  
Vol 10 ◽  
pp. 215517901877507 ◽  
Author(s):  
Yoshiki Nakashima ◽  
Chika Miyagi-Shiohira ◽  
Nana Ebi ◽  
Eri Hamada ◽  
Yoshihito Tamaki ◽  
...  
Keyword(s):  
Survival Rate ◽  
Pancreatic Islets ◽  
Stimulation Index ◽  
Islet Isolation ◽  
University Of Wisconsin Solution ◽  
Porcine Islet ◽  
University Of Wisconsin ◽  
Purification Methods ◽  
Isolation Performance ◽  
Wisconsin Solution

Purification of pancreatic islets is an important step in islet isolation for islet transplantation. In this study, to investigate how a solution composed mainly of Na-lactobionate and histidine (HL) influences the purification of islets, iodixanol was added to a purified solution for porcine islet isolation. A solution (IU) made by adding iodixanol to University of Wisconsin solution and a solution (IHL) made by adding iodixanol to HL solution were used to evaluate the islet isolation performance. We noted no significant differences between the two purification methods with regard to the islet yield, survival rate or purity, score, or stimulation index. These results show that IHL solution is as useful as IU solution for islet purification.

scholarly journals Excellent Islet Yields after 18-h Porcine Pancreas Preservation by Ductal Injection, Pancreas Preservation with MK Solution, Bottle Purification, and Islet Purification Using Iodixanol with UW Solution and Iodixanol with MK Solution

2019 ◽  
Vol 8 (10) ◽  
pp. 1561 ◽  
Author(s):  
Kazuho Kuwae ◽  
Chika Miyagi-Shiohira ◽  
Eri Hamada ◽  
Yoshihito Tamaki ◽  
Kai Nishime ◽  
...  
Keyword(s):  
Stimulation Index ◽  
Islet Isolation ◽  
Preservation Solution ◽  
Porcine Pancreas ◽  
Porcine Islet ◽  
University Of Wisconsin ◽  
Pancreas Preservation ◽  
Purification Methods ◽  
Pancreas Weight ◽  
Wisconsin Solution

Successful islet isolation is the key to successful islet transplantation. Our group recently modified the islet isolation protocol to include pancreatic ductal injection of the preservation solution, pancreas storage in modified extracellular-type trehalose-containing Kyoto (MK) solution, and use of an iodixanol-based purification solution and bottle purification. In this study, we applied these methods to porcine islet isolation after 18-h pancreas preservation and compared two solutions with different compositions in bottle purification. Islet yield before purification was 651,661 ± 157,719 islet equivalents (IE) and 5576 ± 1538 IE/g pancreas weight. An IU solution was made by adding iodixanol to University of Wisconsin solution and an IK solution was made by adding iodixanol to MK solution. The efficacy of the two solutions for islet isolation was compared. There were no significant differences between the two purification methods with regard to islet yield, survival rate, purity, score, or stimulation index. These results indicate that our isolation protocol produces efficient islet yields from prolonged cold-stored pancreas and that IU and IK solutions are equally useful for islet purification.

scholarly journals Pancreatic Islet Purification from Large Mammals and Humans Using a COBE 2991 Cell Processor versus Large Plastic Bottles

2020 ◽  
Vol 10 (1) ◽  
pp. 10
Author(s):  
Hirofumi Noguchi
Keyword(s):  
Shear Force ◽  
Mechanical Damage ◽  
Islet Isolation ◽  
Cell Processor ◽  
Large Mammals ◽  
Purification Step ◽  
Purification Method ◽  
Continuous Density ◽  
Porcine Islet ◽  
Intraportal Infusion

The islet purification step in clinical islet isolation is important for minimizing the risks associated with intraportal infusion. Continuous density gradient with a COBE 2991 cell processor is commonly used for clinical islet purification. However, the high shear force involved in the purification method using the COBE 2991 cell processor causes mechanical damage to the islets. We and other groups have shown human/porcine islet purification using large cylindrical plastic bottles. Shear stress can be minimized or eliminated using large cylindrical plastic bottles because the bottles do not have a narrow segment and no centrifugation is required during tissue loading and the collection processes of islet purification. This review describes current advances in islet purification from large mammals and humans using a COBE 2991 cell processor versus large cylindrical plastic bottles.

Collagenase does Not Persist in Human Islets following Isolation

2012 ◽  
Vol 21 (11) ◽  
pp. 2531-2535 ◽  
Author(s):  
Sarah E. Cross ◽  
Stephen J. Hughes ◽  
Anne Clark ◽  
Derek W. R. Gray ◽  
Paul R. V. Johnson
Keyword(s):  
Human Islets ◽  
Isolation Procedure ◽  
Human Islet ◽  
The Other ◽  
Islet Isolation ◽  
Time Points ◽  
Bacterial Collagenase ◽  
University Of Wisconsin ◽  
Collection Phase ◽  
Wisconsin Solution

Optimal human islet isolation requires the delivery of bacterial collagenase to the pancreatic islet–exocrine interface. However, we have previously demonstrated the presence of collagenase within human islets immediately following intraductal collagenase administration. This potentially has significant implications for patient safety. The present study aimed to determine if collagenase becomes internalized into islets during the isolation procedure and if it remains within the islet postisolation. Islet samples were taken at various stages throughout 14 clinical human islet isolations: during digest collection, following University of Wisconsin solution incubation, immediately postisolation, and after 24 h of culture. Samples were embedded in agar, cryosectioned, and then assessed by immunolabeling for collagenase and insulin. Immunoreactivity for collagenase was not observed in isolated islets in any preparation. Collagenase labeling was detected in one sample taken at the digest collection phase in one islet preparation only. No collagenase-specific labeling was seen in islets sampled at any of the other time points in any of the 14 islet preparations. Collagenase that enters islets during intraductal administration is washed out of the islets during the collection phase of the isolation process and thus does not remain in islets after isolation. This observation alleviates some of the important safety concerns that collagenase remains within islet grafts.

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