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 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 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 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 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 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 Comparison of methods for purification of bacteriophage lysates of gram-negative bacteria for personalized therapy

2021 ◽  
Author(s):  
RB Gorodnichev ◽  
MA Kornienko ◽  
NS Kuptsov ◽  
AD Efimov ◽  
VI Bogdan ◽  
...  
Keyword(s):  
Density Gradient ◽  
Sucrose Gradient ◽  
Sucrose Density Gradient ◽  
Density Gradient Ultracentrifugation ◽  
Phage Lysate ◽  
Purification Method ◽  
Cscl Density Gradient ◽  
Cell Components ◽  
Purification Methods ◽  
Sucrose Gradient Ultracentrifugation

Phage therapy is a promising method of treating antibiotic-resistant infections. To obtain a safe therapeutic formulation, bacterial cell components, including endotoxins, must be removed from the phage lysate. This study was aimed at comparing the efficacy of purification methods for phage lysates intended for therapeutic use. Phages vB_KpnM_Seu621 (Myoviridae) and vB_KpnP_Dlv622 (Autographiviridae) were grown using the KP9068 strain of Klebsiella pneumoniae as a host. The obtained lysates were purified using phage precipitation with polyethylene glycol, CsCl density gradient ultracentrifugation, sucrose density gradient ultracentrifugation, precipitation with 100 kDa centrifugal filter units, and phage concentration on 0.22 µm cellulose filters in the presence of MgSO4. Endotoxin concentrations were determined by LAL testing. The obtained lysates contained 1.25 × 1012 ± 7.46 × 1010 and 2.25 × 1012 ± 1.34 × 1011 PFU/ml of vB_KpnM_Seu621 and vB_KpnP_Dlv622, respectively, and had endotoxin concentrations of 3,806,056 ± 429,410 and 189,456 ± 12,406 EU/ml, respectively. CsCl gradient ultracentrifugation was found to be the optimal conventional purification method in terms of reducing endotoxin concentrations and maintaining phage titers (303 ± 20 — 313 ± 35 EU/ml, 1.5–2.75 × 1012 ± 1.71 × 1011 PFU/ml). Sucrose gradient ultracentrifugation and filtration in the presence of MgSO4 were found to be the optimal non-traditional purification methods. A method for phage lysate purification should be selected for each phage preparation individually. Sucrose gradient ultracentrifugation and filtration in the presence of MgSO4 hold promise as purification methods that can produce phage preparations suitable for intravenous administration.

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.

scholarly journals Method for purification of collagen: A systematic review

2020 ◽  
pp. 99-112
Author(s):  
Nursyahidatul Azwa Awang ◽  
Azura Amid ◽  
Zatul Iffah Arshad
Keyword(s):  
Systematic Review ◽  
High Yield ◽  
Market Demand ◽  
Purification Method ◽  
Affinity Tags ◽  
Advantages And Disadvantages ◽  
Qualitative Systematic Review ◽  
Purification Methods ◽  
Technical Details ◽  
Different Sources

Collagen has diverse applications in various industries and thus a various method of purifications has been developed over the years to produce high yield and high purity of collagen to fulfill collagen market demand. The objective of this qualitative systematic review is to summarize the current literature on the existing purification methods for collagen from different sources. Toward this end, three databases were searched and articles were screened for all original articles explaining the purification of collagen regardless of their main sources. After the comprehensive screening, out of 715 articles, 12 articles were chosen and were further reviewed. The analysis of the technical details, theory, advantages, and disadvantages of the techniques used are reported in this study. We found four types of purification methods that commonly used to purify various types of collagen from mammals, marine, bacteria, and recombinant collagen. The correlations between the prevalence of the method and the efficiency of collagen purification were also identified. It highlighted that many factors should be considered before choosing any method of purification such as types, sources and structure of the collagen itself. Affinity chromatography commonly used for purification of recombinant collagen as they have been genetically modified and appended by affinity tags whereas conventional purification method is preferable for non-recombinant collagen from mammalian and marine sources.

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