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

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.

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

Cell Medicine ◽

10.1177/2155179017733090 ◽

2018 ◽

Vol 10 ◽

pp. 215517901773309 ◽

Cited By ~ 1

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.

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

Cell Medicine ◽

10.3727/215517916x693131 ◽

2017 ◽

Vol 9 (1-2) ◽

pp. 45-51 ◽

Cited By ~ 8

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.

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Notice of forthcoming erratum

The Scientific World JOURNAL ◽

10.1100/tsw.2003.73 ◽

2003 ◽

Vol 3 ◽

pp. 826-826

Keyword(s):

Density Gradient ◽

Islets Of Langerhans ◽

Porcine Pancreas ◽

University Of Wisconsin Solution ◽

Porcine Islet ◽

University Of Wisconsin ◽

Published Paper ◽

Wisconsin Solution

The recently published peer-reviewed protocol article entitled Purification of Islets of Langerhans from Porcine Pancreas by John M. Graham (TheScientificWorldJOURNAL (2002) 2, 1657�1661, ISSN 1537-744X; DOI 10.1100/tsw.2002.847) is being revised. TheScientificWorldJOURNAL will shortly publish the article as an erratum entitled Iodixanol Density Gradient Preparation in University of Wisconsin Solution for Porcine Islet Purification. Links between the published paper and erratum will be made to facilitate cross-referencing.22 August 2003TheScientificWorldJOURNAL

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Comparison of Purification Solutions with Different Osmolality for Porcine Islet Purification

Cell Medicine ◽

10.3727/215517916x693140 ◽

2017 ◽

Vol 9 (1-2) ◽

pp. 53-59 ◽

Cited By ~ 9

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.

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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

Journal of Clinical Medicine ◽

10.3390/jcm8101561 ◽

2019 ◽

Vol 8 (10) ◽

pp. 1561 ◽

Cited By ~ 3

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.

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

Journal of Clinical Medicine ◽

10.3390/jcm10010010 ◽

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.

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Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins

Scientific Reports ◽

10.1038/s41598-021-84682-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Alexander Pralow ◽

Alexander Nikolay ◽

Arnaud Leon ◽

Yvonne Genzel ◽

Erdmann Rapp ◽

...

Keyword(s):

Zika Virus ◽

Animal Cell ◽

Gradient Centrifugation ◽

Viral Envelope ◽

Protein Glycosylation ◽

High Yield ◽

Structural Protein ◽

Site Specific ◽

E Protein ◽

The Impact

AbstractHere, we present for the first time, a site-specific N-glycosylation analysis of proteins from a Brazilian Zika virus (ZIKV) strain. The virus was propagated with high yield in an embryo-derived stem cell line (EB66, Valneva SE), and concentrated by g-force step-gradient centrifugation. Subsequently, the sample was proteolytically digested with different enzymes, measured via a LC–MS/MS-based workflow, and analyzed in a semi-automated way using the in-house developed glyXtoolMS software. The viral non-structural protein 1 (NS1) was glycosylated exclusively with high-mannose structures on both potential N-glycosylation sites. In case of the viral envelope (E) protein, no specific N-glycans could be identified with this method. Nevertheless, N-glycosylation could be proved by enzymatic de-N-glycosylation with PNGase F, resulting in a strong MS-signal of the former glycopeptide with deamidated asparagine at the potential N-glycosylation site N444. This confirmed that this site of the ZIKV E protein is highly N-glycosylated but with very high micro-heterogeneity. Our study clearly demonstrates the progress made towards site-specific N-glycosylation analysis of viral proteins, i.e. for Brazilian ZIKV. It allows to better characterize viral isolates, and to monitor glycosylation of major antigens. The method established can be applied for detailed studies regarding the impact of protein glycosylation on antigenicity and human pathogenicity of many viruses including influenza virus, HIV and corona virus.

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Pancreatic islet transplantation under the kidney capsule of mice: model of refinement for molecular and ex-vivo graft analysis

Laboratory Animals ◽

10.1177/00236772211004051 ◽

2021 ◽

pp. 002367722110040

Author(s):

Julien Thévenet ◽

Valery Gmyr ◽

Nathalie Delalleau ◽

François Pattou ◽

Julie Kerr-Conte

Keyword(s):

Islet Transplantation ◽

Pain Treatment ◽

Ex Vivo ◽

Well Being ◽

Diabetic Patients ◽

Mice Model ◽

Pancreatic Islet Transplantation ◽

Post Transplantation ◽

Kidney Capsule

Diabetes cell therapy by human islet transplantation can restore an endogenous insulin secretion and normal glycaemic control in type 1 diabetic patients for as long as 10 years post transplantation. Before transplantation, each clinical islet preparation undergoes extensive in-vitro and in-vivo quality controls. The in-vivo quality control assay consists of transplanting human islets under the kidney capsule of immunocompromised mice. Currently, it is considered the best predictive factor to qualify clinical transplant efficiency. This chimeric model offers a wide area of study since it combines the possibility of producing not only quantitative but also a maximum of qualitative data. Today’s technological advances allow us to obtain more accurate and stronger data from the animals used in research while ensuring their comfort and well-being throughout the protocol, including cage enrichment and pain treatment during and after surgery. As demonstrated in this valuable model, we are able to generate more usable results (Refine), while reducing the number of animals used (Reduce), by focusing on the development of ex-vivo analysis techniques (Replace), which clearly highlights the Burch and Russell 3Rs concept.

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Clinical Relevance of Corticosteroid Withdrawal on Graft Histological Lesions in Low-Immunological-Risk Kidney Transplant Patients

Journal of Clinical Medicine ◽

10.3390/jcm10092005 ◽

2021 ◽

Vol 10 (9) ◽

pp. 2005

Author(s):

Domingo Hernández ◽

Juana Alonso-Titos ◽

Teresa Vázquez ◽

Myriam León ◽

Abelardo Caballero ◽

...

Keyword(s):

Kidney Transplant ◽

Rejection Rate ◽

Open Label ◽

Post Transplantation ◽

Protocol Biopsy ◽

Corticosteroid Withdrawal ◽

Transplant Patients ◽

Prospective Phase ◽

Kidney Transplant Patients ◽

The Impact

The impact of corticosteroid withdrawal on medium-term graft histological changes in kidney transplant (KT) recipients under standard immunosuppression is uncertain. As part of an open-label, multicenter, prospective, phase IV, 24-month clinical trial (ClinicalTrials.gov, NCT02284464) in low-immunological-risk KT recipients, 105 patients were randomized, after a protocol-biopsy at 3 months, to corticosteroid continuation (CSC, n = 52) or corticosteroid withdrawal (CSW, n = 53). Both groups received tacrolimus and MMF and had another protocol-biopsy at 24 months. The acute rejection rate, including subclinical inflammation (SCI), was comparable between groups (21.2 vs. 24.5%). No patients developed dnDSA. Inflammatory and chronicity scores increased from 3 to 24 months in patients with, at baseline, no inflammation (NI) or SCI, regardless of treatment. CSW patients with SCI at 3 months had a significantly increased chronicity score at 24 months. HbA1c levels were lower in CSW patients (6.4 ± 1.2 vs. 5.7 ± 0.6%; p = 0.013) at 24 months, as was systolic blood pressure (134.2 ± 14.9 vs. 125.7 ± 15.3 mmHg; p = 0.016). Allograft function was comparable between groups and no patients died or lost their graft. An increase in chronicity scores at 2-years post-transplantation was observed in low-immunological-risk KT recipients with initial NI or SCI, but CSW may accelerate chronicity changes, especially in patients with early SCI. This strategy did, however, improve the cardiovascular profiles of patients.

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