Euro-Collins Solution Research Articles

An efficient polymer cocktail-based transportation method for cartilage tissue, yielding chondrocytes with enhanced hyaline cartilage expression during in vitro culturing

BackgroundChondrocytes are used in cell-based therapies such as autologous chondrocyte implantation (ACI) and matrix-associated cartilage implantation (MACI). To transport the cartilage tissue to the laboratory for in vitro culturing, phosphate-buffered saline (PBS), Euro-Collins solution (ECS) and Dulbecco's Modified Eagle's Medium (DMEM) are commonly employed at 4–8 °C. MethodsIn this study, eight samples of human cartilage biopsy tissues from elderly patients with severe osteoarthritis undergoing arthroscopy, which would otherwise have been discarded, were used. The cartilage tissue samples were compared to assess the cell yield between two transportation groups: i) a thermo-reversible gelation polymer (TGP) based method without cool preservation (∼25 °C) and ii) ECS transport at 4 °C. These samples were subjected to in vitro culture in a two-dimensional (2D) monolayer for two weeks and subsequently in a three-dimensional (3D) TGP scaffold for six weeks. ResultsThe cell count obtained from the tissues transported in TGP was higher (0.2 million cells) than those transported in ECS (0.08 million cells) both after initial processing and after in vitro culturing for 2 weeks in 2D (18 million cells compared with 10 million cells). In addition, mRNA quantification demonstrated significantly higher expression of Col2a1 and SOX-9 in 3D-TGP cultured cells and lower expression of COL1a1 in RT-PCR, characteristic of the hyaline cartilage phenotype, than in 2D culture. ConclusionThis study confirms that the TGP cocktail is suitable for both the transport of human cartilage tissue and for in vitro culturing to yield better-quality cells for use in regenerative therapies.

Effects of the preservation medium and storage duration of domestic cat ovaries on the maturational and developmental competence of oocytes <i>in vitro</i>

We examined the effectiveness of saline, Euro-Collins solution (EC), and ET-Kyoto solution (ET-K) as preservation media for the cold storage of feline ovaries. Ovaries were maintained in these media at 4°C for 24, 48, or 72 h until oocyte retrieval. The ET-K group exhibited a higher oocyte maturation rate than the saline group after 72 h of storage. Moreover, ET-K could sustain the competence of the feline oocytes to cleave after 48 h, and the morula formation rate of the ET-K group was higher than that of the other groups after 24 and 48 h. Furthermore, the ET-K group exhibited a higher blastocyst formation rate than the other groups after storage for 24 h, and only ET-K retained the developmental competence in blastocysts after 48 h of storage. In addition, regarding the cell numbers of the blastocysts, there was no significant difference among the tested groups. In conclusion, our results indicate that ET-K is a suitable preservation medium for feline ovaries.

In Vitro Culture Expansion and Characterization of Buccal Mucosal Epithelial Cells for Tissue Engineering Applications in Urethral Stricture After Transportation Using a Thermoreversible Gelation Polymer.

Introduction: The transportation of tissues from hospitals to clinical laboratories for cell therapy is an essential component of regenerative medicine. Previously, we used laboratory-cultured mucosal cells from buccal epithelium expanded and encapsulated using a scaffold-hybrid approach to the urethral stricture (BEES-HAUS) procedure. In this study, to improve the outcomes, we compared the thermoreversible gelation polymer (TGP) transportation procedure with conventional culture methods, and reported its advantages. Methods: Human buccal mucosal tissues in Phase I of the study were transported in Euro-Collins solution (ECS) and the cells obtained were cultured in two-dimensional (2D) Dulbecco's modified Eagle's medium (DMEM), CnT-Prime epithelial 2D differentiation medium (CnT-PR), and a three-dimensional (3D)-TGP scaffold. In Phase II, tissues were transported in a TGP cocktail and the ECS. The cells were cultured in 2D-DMEM and 3D-TGP, quantified, and characterized by immunohistochemistry. Results: The cells in 3D-TGP culture maintained epithelial morphology in a better manner compared with 2D-DMEM, in which they developed fibroblast-like morphology. The TGP-transported cells grew rapidly. Immunohistochemical analysis results for AE1/AE3, EGFR, integrin-β1, p63, and p75 were intensely positive in 3D-TGP. Conclusion: The TGP-based cocktail used in human buccal tissue transportation yielded cells with better morphology maintenance. The TGP scaffold provides an optimal in vitro environment wherein epithelial cells better maintain their native phenotype compared to those cultured through conventional methods. These results suggest using TGP for the transportation and culture of human buccal tissues for clinical applications. In addition, the use of a TGP-based cocktail for the transport of other tissues for regenerative medicine applications is worth further analysis.

Techniques of orthotopic renal transplantation. II. Size-matched porcine grafts in monkey recipients.

ABSTRACTPurposeAs a classical xenotransplantation model, porcine kidneys have been transplanted into the lower abdomen of non-human primates. However, we have improved upon this model by using size-matched grafting in the orthotopic position. The beneficial aspects and surgical details of our method are reported herein.MethodsDonors were two newborn pigs (weighting 5 to 6 kg) and recipients were two cynomolgus monkeys (weighting, approximately, 7 kg). After bilateral nephrectomy, kidneys were cold-transported in Euro-Collins solution. The porcine kidney was transplanted to the site of a left nephrectomy and fixed to the peritoneum.ResultsKidneys transplanted to the lower abdomen by the conventional method were more susceptible to torsion of the renal vein (two cases). In contrast, early-stage blood flow insufficiency did not occur in orthotopic transplants of theleft kidney.ConclusionsSize-matched porcine-primate renal grafting using our method of transplanting tothe natural position of the kidneys contributes to stable post-transplant blood flow to the kidney.

Resection of an only kidney with isolated perfusion by cold EuroCollins solution

Resection of an only kidney with isolated perfusion by cold EuroCollins solution

Combination of Euro-Collins and University of Wisconsin Solution: An Effective and Economic Substitute for Organ Procurement

PurposeMany types of preservation fluid were used in liver procurement. Undoubtedly, the gold standard is the University of Wisconsin (UW) solution. But the solution is expensive. The aim of this study was to evaluate the results of combined acetated Ringer solution, Euro-Collins solution, and UW solution. Materials and MethodsAll patients undergoing adult liver transplantation from cadaveric donor during January 2013 to December 2017 in King Chulalongkorn Memorial Hospital were included in this study. Donor and recipient characteristics, preservation fluid, operative data, and postoperative outcomes were recorded. ResultsA total of 102 patients receiving liver transplants were enrolled into the study. The mean age of donors was 34.2 years. The mean total ischemic time was 420.93 minutes. In recipients, posttransplantation complications were the following: (1) primary nonfunction in 1 patient (0.98%); (2) early allograft dysfunction in 23 patients (22.5%); (3) hepatic artery thrombosis in 3 patients (2.7%); (4) hepatic venous outflow obstruction in 2 patients (1.96%); (5) biliary leakage in 1 patient (0.98%); (6) biliary anastomosis stenosis in 4 patients (3.92%); and (7) biliary nonanastomosis stenosis in 1 patient (0.98%). No inhospital mortality was occurred. Overall mortality rate is 7.8% (8/102). One-, 3-, and 5-year survival were 95.9%, 91.5%, and 88.4%, respectively. ConclusionsThe combination of acetated Ringer solution, Euro-Collins solution, and UW solution is effective and economic for liver preservation. Further study should be conducted.

Cardiac myocyte functional and biochemical changes after hypothermic preservation in vitro. Protective effects of storage solutions.

In this study, we evaluated cardiac myocyte viability and function under hypothermic conditions with four types of storage solutions, saline solution, Euro-Collins solution, University of Wisconsin solution, and MCDB 107 medium. Cardiac myocytes were isolated from neonatal rat ventricles by collagenase dispersion and cultured for 4 days with MCDB 107 medium. A total of 12.5 x 10(5) myocytes per culture dish was used and the myocytes were incubated at 4 degrees C for 6, 12, 18, and 24 hours in the various storage solutions. After each incubation time, creatine kinase and lactate dehydrogenase were measured in the storage solutions. The myocytes were then incubated for 24 hours at 37 degrees C to evaluate the recovery of the myocyte beating rate. In the MCDB 107 group (n = 7), the recovery ratio of myocyte beating rate was complete by 12 hours, then decreased to 44.8% of control (beating rate before hypothermic incubation) at 24 hours. The saline, Euro-Collins, and University of Wisconsin groups (n = 7 each) had significantly lower recovery ratios than the MCDB 107 group (at 12 hours: 61.0%, 32.2%, and 48.9%; at 18 hours: 0.0%, 5.5%, and 15.1% of control, respectively). Release of creatine kinase and lactate dehydrogenase in the MCDB 107 group gradually increased and at 24 hours was 143.2 mIU/flask and 486.2 mIU/flask, respectively. However, the saline and University of Wisconsin groups had significantly increased creatine kinase and lactate dehydrogenase values at 24 hours (creatine kinase: 334.6 and 319.6 mIU/flask; lactate dehydrogenase: 821.6 and 654.4 mIU/flask, respectively). The Euro-Collins group showed the greatest increase in both markers (creatine kinase: 1587.5, lactate dehydrogenase: 2106.9 mIU/flask). In summary, saline and University of Wisconsin solutions showed a beneficial effect on recovery of myocyte viability at 12 hours compared with Euro-Collins solution, however MCDB 107 medium had the best overall protective effect on cultured myocytes. Accordingly, alternate hypothermic storage solutions, such as cell-culture medium, may have protective characteristics that are suitable for cardiac preservation.

Improving the viability of tissue-resident stem cells using an organ-preservation solution.

Human clinical specimens are a valuable source of tissue‐resident stem cells, but such cells need to be collected immediately after tissue collection. To extend the timescale for collection from fresh human samples, we developed a new extracellular fluid (ECF)‐type preservation solution based on a high‐sodium and low‐potassium solution containing low‐molecular‐weight dextran and glucose, which is used for preservation of organs for transplantation. In this study, we compared the preservation of tissue‐resident stem cells using our ECF solution with that using three other solutions: PBS, Dulbecco’s modified Eagle’s medium and Euro‐Collins solution. These solutions represent a common buffer, a common culture medium and a benchmark organ‐preservation solution, respectively. Lung tissues were removed from mice and preserved for 72 h under low‐temperature conditions. Of the solutions tested, only preservation in the ECF‐type solution could maintain the proliferation and differentiation capacity of mouse lung tissue‐resident stem cells. In addition, the ECF solution could preserve the viability and proliferation of human alveolar epithelial progenitor cells when stored for more than 7 days at 4 °C. The mean viability of human alveolar type II cells at 2, 5, 8 and 14 days of low‐temperature preservation was 90.9%, 84.8%, 85.7% and 66.3%, respectively, with no significant differences up to 8 days. Overall, our findings show that use of our ECF‐type preservation solution may maintain the viability and function of tissue‐resident stem cells. Use of this preservation solution may facilitate the investigation of currently unobtainable human tissue specimens for human stem cell biology.

Low-dose In Situ Perfusion With Euro-Collins Solution Is Effective for the Procurement of Marginal Kidney Grafts From Donation After Circulatory Death Donors

We have adopted a modified method to resuscitate kidneys from donation after circulatory death (DCD) donors with the use of Euro-Collins (EC) solution instead of University of Wisconsin solution. This study aimed to evaluate kidney transplantation (KTx) outcomes of DCD procured with low-dose in situ perfusion using EC solution. Patients and MethodsKTx was performed in 8 adults. Kidney grafts were procured following in situ perfusion with approximately 1 L of EC solution and preserved in the solution. The kidney donor profile index value was 88% ± 21%. The terminal creatinine level of the donors was 5.5 ± 3.4 mg/dL. Of the 8 donors, 6 experienced oligoanuria prior to graft procurement. ResultsThe mean age of the recipients and the hemodialysis vintage were 50 ± 10 years and 161 ± 25 months, respectively. The warm and cold ischemic times were 8.3 ± 7.9 minutes and 8.7 ± 4.3 hours, respectively. All grafts functioned after a delayed graft function of 10.6 ± 6.9 days (2–25 days). There was neither immediate graft function nor primary nonfunction. The patient and graft survivals were both 100% with a terminal creatinine level of 1.3 ± .5 mg/dL. ConclusionsKidney grafts procured from DCD donors with a high kidney donor profile index value demonstrated good renal function with an excellent midterm outcome. Low-dose in situ perfusion with EC solution is effective for the procurement of marginal kidney grafts from DCD donors under optimal conditions such as a relatively shorter preservation time.

Гетеротопическая ксенотрансплантация фрагментов аорты новорожденных поросят после гипотермической инкубации в консервирующих растворах

Nowadays the porcine vessels and heart valves have been widely applied in transplantology. Due to a high level of Gal-α-1,3-Gal (α-Gal epitope) expression, the porcine bioprostheses are specially treated prior to transplantation either to remove this epitope or to deactivate it. Our previous findings showed that certain media, used for hypothermic storage (HS), reduced the α-Gal epitope expression in aortic tissue in vitro. Here, we studied the HS impact in the histidine-tryptophan-ketoglutarate (HTK), Euro-Collins and Dulbecco’s Modified Eagle Medium (DMEM) solutions on aortic graft integrity in vivo. The analysis of aortic wall histological structure and the evaluation of the immunoproteasome subunits LMP2 and LMP7 expression level demonstrated the aortic fragments, subjected to HS in Euro-Collins solution to day 7 after xenotransplantation to cause a less pronounced immune response as compared to the HTK- and DMEM-incubated xenografts. This may be partially due to the Euro-Collins medium composition. Our findings may be of interest for ela-borating the novel methods for donor material pre-transplantation preparation. Probl Cryobiol Cryomed 2019; 29(1): 073–087

Fortification of Preservation Solution With Nitroprusside Does Not Alter Lung Allograft Survival in Clinical Human Lung Transplantation.

Background: Nitric oxide improves gas exchange following primary lung allograft dysfunction. Nitroprusside, a potent nitric oxide donor, has reduced reperfusion injury and improved oxygenation in experimental lung transplantation. Methods: We sought to study the effect on lung allograft outcomes of fortifying the preservation solution with nitroprusside. We conducted a single-center clinical study of 46 consecutive lung recipients between 1998 and 2000: 24 patients received donor organs preserved in modified Euro-Collins solution with prostaglandin E1 (PGE1) (control group), and 22 patients received organs preserved in modified Euro-Collins with PGE1 and nitroprusside (NP group). The primary endpoint was overall survival. Results: Baseline characteristics were similar between the groups except for a significantly longer graft ischemic time in the NP group vs the control group (253.3 ± 52 vs 225.3 ± 41 minutes, respectively, P=0.04). No significant differences were found in partial pressure arterial oxygen to fraction inspired oxygen ratio at ≤48 hours, primary graft dysfunction, or bronchiolitis obliterans-free days. Overall survival at 1, 3, and 5 years was 89%, 73%, and 63% in the control group and 76%, 38%, and 23% in the NP group. Log-rank survival analysis showed that the NP group had a significantly increased risk of mortality (P=0.034) compared to the control group. Conclusion: The addition of nitroprusside to the lung transplant perfusate in this clinical trial did not improve survival; however, a large randomized trial would likely reduce confounding ischemia times and increase the power of the study.

Re: Retrogradein situversus antegrade pulmonary preservation in clinical lung transplantation: a single-centre experience

Historically, grafts were preserved by means of autoperfusion with extracorporeal circulation, topical cooling or donor core cooling. In the 1980s, the standard lung preservation method was single pulmonary artery flush perfusion, using modified Euro-Collins solution at 4°C [1]. However, reperfusion injury remained a significant factor for morbidity and early mortality after lung transplant. Since the early years of lung preservation strategies, some advances have emerged in transplant teams, such as adding prostaglandin E2 to prevent counteract temperature and potassium-induced vasoconstriction. The use of low potassium dextran glucose solutions shows better overall lung function, superior early oxygenation, higher lung compliance, lower incidence of severe primary graft dysfunction and 30-day mortality [2]. As Gohrbandt et al. point out, the route to administer the preservation solution may also have an impact on early lung graft performance [3]. Anterograde flush initiating the infusion through the pulmonary artery with pulmonary vein drainage was the traditional approach for many years [4]. This technique, however, presents a number of limitations. Flushing the pulmonary artery leaves the bronchial tissue to be preserved by topical cooling alone, raising the complication from the bronchial suture. Also, clots and fat or brain tissue emboli, especially in donors with major trauma and multiple bone fractures, may hamper uniform flushing and cooling and, therefore, impair graft function upon reperfusion. Adding a retrograde flush, whereby the preservation solution is administered via the left atrial appendage or four pulmonary veins into the pulmonary venous system and drained through the pulmonary artery, has the potential to flush not only pulmonary vessels, but also the bronchial network, contributing to the adequate perfusion of the tracheobronchial submucosa tissue, and also limiting the effect of hypothermic pulmonary arterial vasoconstriction. On the other hand, less ventilated areas, such as the posterior segments of lower lobes, are better flushed. Also residual blood and harmful constituents and any possible microthrombi that may obstruct the pulmonary vessels are washed out. The evidence from clinical series of this technique is limited [5]; authors such as Gohrbandt et al. show us that there are no clinical benefits of retrograde lung preservation alone and that it may cause or prolong mechanical ventilation and primary graft dysfunction at 72 h and prolong ICU stay [3]. On the other hand, some evidence has been published, showing that biphasic lung preservation is not only clinically feasible [6], but also related with better early oxygenation ratio, lower mean airway pressure and chest radiography score [7, 8]. Many transplant teams have now adopted a biphasic technique with an antegrade flush through the pulmonary artery followed by a retrograde flush through each of the pulmonary veins after heart excision with the lung still ventilated inside the cadaver. However, there is lack of controlled randomized studies.

C-20: Tissue vitrification

Application of the original tissue vitrification method developed by Fahy and used by my research group for rabbit blood vessels and articular cartilage has evolved over time. The formulation was 2.21 M propylene glycol, 3.10 M formamide and 3.10 M dimethylsulfoxide in EuroCollins solution and it was employed with multiple addition/removal steps to minimize osmotic damage to the cells. Ice-free cryopreservation using VS55, as originally described, is limited to small or thin samples where relatively rapid cooling and warming rates are possible. VS55 cryopreservation typically provides excellent extracellular matrix preservation with approximately 80% cell viability and tissue function compared with fresh untreated tissues. In contrast, ice-free cryopreservation using higher concentrations of the same cryoprotectants in EuroCollins solution has several advantages. High viability can be achieved for human and porcine articular cartilage employing an 83% formulation, VS83, in combination with multiple addition/removal steps. Lower cryoprotectant concentrations are not effective for large mammal articular cartilage. VS83 can also be used for tissue matrix preservation with retention of material properties at the expense of cell viability. Addition and removal of the cryoprotectants can be done in a single step followed by washing to minimize residual chemical concentrations. This protocol minimizes tissue cell viability and also reduces tissue immunogenicity suggesting that this vitrification strategy may be an alternative to the decellularization methods being employed for engineering tissues for medical implants. The evolution of these vitrification protocols will be reviewed drawing upon research employing natural and engineered tissues. Disclosure: Author was or is employed by Cell & Tissue Systems, Inc.

Impact of Hypothermic Preservation on Tissue Yield and Viability in Pig Pancreata

IntroductionChronic shortage of quality human cadaveric pancreata limits islet transplantation. Porcine islet xenotransplantation is being explored to increase the donor pool. For clinical-ready islets, centralized animal husbandry, Current Good Manufacturing Practice–regulated processing facilities, and organ transportation support are required. Amount of cold ischemia time (CIT) before isolation significantly affects transplantation. The goal of this study was to determine the maximum safe CIT of whole pancreata before islet isolation. Materials and MethodsPancreata were rapidly removed from Yorkshire pigs (age, 14–22 days) and stored in modified University of Wisconsin solution or in EuroCollins solution at 4°C. Pancreata were processed with <1 hour CIT (control) or stored for 4 or 12 hours before isolation. Islet yield and percent purity and viability were determined after 7 days of in vitro tissue culture and maturation. Samples from nonprocessed pancreata were collected and snap-frozen in liquid nitrogen at 0, 3, 6, 9, 12, 15, and 24 hours of preservation, then analyzed for adenosine diphosphate/adenosine triphosphate ratio as a measure of tissue energetics. ResultsUp to 12 hours in cold storage had no significant impact on overall islet yield after 7 days of in vitro culture compared with controls; islet yield at the end of the maturation process was 28,700 ± 500 islet equivalents per pancreas (mean ± SEM control yield, 30,300 ± 900 islet equivalents per pancreas); islet purity was 75 ± 5% compared with 74 ± 5% in controls. Islet viability was significantly reduced at 12 hours compared with controls (80 ± 6% vs 96 ± 5%; P < .05). The tissue adenosine diphosphate/adenosine triphosphate ratio was maintained within the first 6 hours (1.6 ± 0.1 to 1.8 ± 0.2; P = NS) but was markedly increased during the 24-hour study (3.3 ± 0.1 at 24 hours), indicating a progressive loss of adenosine triphosphate tissue stores. ConclusionsYoung pig pancreata can be hypothermically stored for up to 12 hours without affecting islet yield and purity; however, islet viability is reduced. These data highlight the need for uniform shipping parameters to standardize islet quality, ideally with CIT <6 hours.

Comparative efficacy of Belzer or Euro-Collins solutions for pancreatic preservation during cold ischemic storage in rats

To compare the efficacy of different types of solutions (Belzer or Euro-Collins) for the preservation of rat pancreas during cold ischemia. Thirty Wistar rats were divided into three groups according to the perfusion or storage solution: Group E (perfusion and storage in Euro-Collins solution); Group B (perfusion and storage in Belzer solution) and Group BE (Perfusion in Belzer solution and storage in Euro-Collins solution). After perfusion, the pancreas was excised and stored at 4˚C for 18 hours. Amylase was measured at 6, 12 and 18h, and histological analysis of the pancreas was performed after 18h of cold storage. Amylase was elevated and comparable in Groups E and BE after 12 and 18 hours of ischemia (p<0.05). In the exocrine pancreas, histological differences in the amount of necrosis (p=0.049), lymphocytic infiltrate (p<0.001) and neutrophilic infiltrate (p=0.004) were observed, with more favorable features present in Group B. In the endocrine pancreas, Group B showed less edema (p<0.001), but other parameters were similar among all groups. The Euro-Collins solution is inferior to the Belzer solution for the preservation of rat pancreas during cold ischemia.

Protective Effect of Ginsenoside Rb1 Mixing with Several Lung Flush Solutions on Pulmonary Ischemia-Reperfusion Injury

Objective: To evaluate the protective effects of ginsenoside Rb1 mixed with LPD compared to mixed with several other lung flush solutions on ischemia-reperfusion injury. Method: Three group of rabbit lungs were perfused with three kinds of mixtures of ginsenoside Rb1 with blood, Euro-Collins solution(EC) or low-potassium-dextran solution(LPD) respectively, then lung dry/wet weight ratio and malondiadehyde(MAD) were examined and histological changes were observed. Result: Lung dry/wet weight ratio of LPD and Rb1 group was higher than that of EC and Rb1 and blood and Rb1 groups, whereas MAD of LPD and Rb1 group was significantly less than that of EC and Rb1 and blood and Rb1 groups（P&lt;0.05）. Histological findings showed less damage in LPD and Rb1 group. Conclusion: Ginsenoside Rb1 mixed with LPD shows a better protective effect on pulmonary ischemia-reperfusion injury compared with other mixtures.

Attenuation of Lung Ischemia-Reperfusion Injury by Rho-Associated Kinase Inhibition in a Rat Model of Lung Transplantation

A signaling pathway of the small GTPase Rho and Rho-associated coiled-coil-forming protein kinase (ROCK), regulates the contraction of endothelial cells. We studied the effects of Y-27632, a specific ROCK inhibitor, to clarify the role of Rho/ROCK in the pathogenesis of ischemia-reperfusion lung injury in a rat model of single-lung transplantation (LTX). We flushed 5 donor rat lungs with Euro-Collins solution, and 5 donor lungs with Euro-Collins + Y-27632, 0.03 mg/ml, and preserved the lungs for 6 h at 4°C before reperfusion for 4 h. The 5 rat recipients of Y-27632-treated lungs also received a 10-mg/kg bolus of Y-27632 i.p. 30 min before reperfusion. Pretreatment of the donor lungs and recipient rats with Y-27632 prominently suppressed the post-LTX edema, while the permeability index was only slightly decreased. The (1) numbers of neutrophils and macrophages, and (2) tumor necrosis factor (TNF)-α concentration, were significantly lower in the bronchoalveolar lavage fluid of treated than untreated lungs. Y-27632 (1) inhibited the migration of inflammatory cells into the alveolar space, (2) decreased the production of TNF-α, and (3) attenuated the edema after LTX. Endothelial Rho and ROCK may play an important role in the pathogenesis of post-LTX injury.

Review: Macro-Encapsulation of Islets in Polyvinyl Alcohol Hydrogel

Diabetes mellitus (DM) can be cured by adequate insulin secretion from a relatively small volume of cells. Cell encapsulation enables allo- and even xeno-geneic cell therapy without immunosuppression. Micro-encapsulated islets used in recent clinical trials are not fully retrievable after transplantation. This paper summerizes the development of retrievable and theoretically replaceable macro-encapsulated islets using polyvinyl alcohol (PVA) hydrogel. An aqueous solution of PVA becomes a gel through micro crystallization by freezing and thawing. Utilizing this feature, PVA-macro-encapsulated islets (PVA-MEIs) were developed. Rat islets suspended in Euro-Collins solution containing 3% PVA were encapsulated in a mesh-reinforced PV A hydrogel sheet by freezing and thawing. The feasibility of PVA-MEIs for DM therapy was tested in vitro and in vivo. PVA-MEIs showed glucose-responsive insulin secretion in vitro even after 14-day culture. Rat PVA-MEIs cultured in media containing fresh human plasma showed no morphological changes and maintained insulin content. Intra-peritoneal transplantation of PVA-MEIs containing 750 rat islets ameliorated hyperglycemia in streptozotocin (STZ)-induced diabetic mice to nearly normal levels for up to 30 days with a consistent increase in body weight. Transplantation of PVA-MEIs also prevented metabolic and renal disorders in STZ-induced diabetic mice. PVA-MEIs cryo-preserved for 1, 7, and 30 days showed similar function in vitro and corrected hyperglycemia after intra-peritoneal transplantation in STZ-induced diabetic mice. Intra-peritoneal transplantation of PVA-MEIs containing iso- or allo-geneic islets (approx. 2,000 islets) ameliorated hyperglycemia in STZ-induced diabetic rats in a similar manner for almost half a year although the efficacy gradually decreased with time. Transplantation of PVA-MEIs ameliorated hyperglycemia in diabetic mice and rats without immunosupression. Retrievable and theoretically replaceable PVA-MEIs that can secure cell entrapment can mitigate the potential risks associated with xeno-geneic cells and cells made from undifferentiated cells. Therefore, PVA-MEIs are a promising modality for future DM therapy.

Evaluation of Liver Preservation Solutions by Using Rats Transgenic for Luciferase

IntroductionThe solution in which graft tissue is stored (that is, preservation solution) is an important component of liver transplantation technology. Its protective effect is induced by substances in the solution, including radical scavengers, buffers, and energy-giving substances. New preservation solutions have proven to be effective in preventing organ damage during cold ischemia and in extending the time limits for storage. AimThis study determined the relationship between luminescence intensity and content of adenosine triphosphate (ATP) in liver tissue and proposes a new ex vivo screening system that uses Lewis rats transgenic for luciferase for evaluating the effectiveness of preservation solutions. MethodsSamples (diameter, 2 mm) of liver were obtained from transgenic rats. The viability of these tissues after storage for as long as 6 hours in University of Wisconsin (UW) solution, extracellular trehalose solution of Kyoto, Euro-Collins (EC) solution, histidine–tryptophan–ketoflutarate solution, low potassium dextran solution, or normal saline was assessed by determining ATP content and luminescence intensity. ResultsLuminescence had a linear relationship (R = 0.88) with ATP levels. Regardless of the preservation solution used, the luminescence intensities of the liver tissue chips decreased linearly with time especially through a short span of time (0 to 2 hours; R2 = 0.58–1.0). The luminescence of liver chip tissues maintained long term (2 to 6 hours) in UW solution tended to be higher than those of tissues stored in other solutions (P < .05; 6 hours). On the basis of luminescence intensity, EC might be preferable to the other solutions tested for ultra-short–term storage (0.5 to 2 hours). ConclusionOur model, which combines the use of the bioimaging system and Lewis rats transgenic for luciferase, effectively assessed the viability of liver tissue samples. We believe that this ex vivo screening system will be an effective tool for evaluating preservation solutions for liver grafts.

46 HYPOTHERMIC STORAGE FOR 10 DAYS OF BOVINE EMBRYOS USING TYPE III ANTIFREEZE PROTEIN

Recently, we developed a medium that enabled bovine embryos to be held for up to 7 days at 4°C (Tsuchiya et al. 2014 IETS meeting). To be of practical value, mammalian embryo preservation at hypothermic temperatures must be able to maintain viability for periods longer than 7 days. Antifreeze proteins (AFP) were discovered in various organisms (such as fish, insects, plants, and bacteria) living in cold regions. They show a unique ability to protect cold-sensitive cells from hypothermic damage. Here, we found that a biomolecule known as type III AFP solubilised into an optimized solvent can keep alive bovine embryos for a maximum of 10 days at hypothermic temperatures. First, human hepatoma (HepG2) and rat insulinoma (RIN-5F) cells were stored at 4°C in Euro-Collins solution (Kobayashi Seiyaku, Osaka, Japan) supplemented with or without 10 mg mL–1 of type III AFP for 24 h. The viability rate of the cells was assessed by trypan blue (Dojindo, Kumamoto, Japan) dissolved in PBS. Second, high-quality blastocysts produced in vivo were stored at 4°C in a plastic ministraw in 25 mM HEPES medium 199 plus 20% fetal bovine serum (FBS) supplemented with or without 10 mg mL–1 of type III AFP for 10 days. Following hypothermic preservation, the chilled embryos were squeezed out of the straw into PBS and washed 3 times in the same medium. Subsequently, the embryos were cultured in CR1aa medium supplemented with 5% FBS for 48 h at 38.5°C under 5% CO2 in air with high humidity. The viability and hatching rate of the embryos were assessed at the end of the culture period. Finally, 4 embryos stored for 10 days with type III AFP were reloaded into plastic straws with the washing medium and transferred into recipient heifers (1 embryo per recipient). Pregnancy was determined by real-time B-mode ultrasonography (Convex scanner HS-1500, Honda Electronics Co. Ltd., Toyohashi, Japan) on Day 60 of gestation. Data were analysed using chi-squared and Student's t-tests. In the absence of type III AFP, the cell viability values of HepG2 and RIN-5F after hypothermic storage for 24 h were only 5 and 22%, respectively. However, in the presence of type III AFP, the cell viability values were dramatically increased (HepG2: 71%; RIN-5F: 59%) than those measured without type III AFP. To examine the effect of type III AFP for hypothermic preservation of bovine embryos, we used 80 high-quality embryos produced in vivo and assigned them randomly to 2 experimental groups. The viability and hatching rates of the chilled embryos stored with type III AFP for 10 days were significantly higher (58 and 30%, respectively) than those of without type III AFP (28 and 0%, respectively). The pregnancy rate of the chilled embryos stored with type III AFP was 50%: 2 pregnancies continue until now. In conclusion, prolongation of short-term preservation period with AFP-containing fluid will realise LN2-free storage of bovine embryos for a 10-day period. This work was supported by the Program for Promotion of Basic and Applied Research for Innovations in Bio-Oriented Industry.