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The origins of low-temperature tissue storage research date back to the late 1800s. Over half a century later, osmotic stress was revealed to be a main contributor to cell death during cryopreservation. Consequently, the addition of cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), or propylene glycol (PG), although toxic to cells at high concentrations, was identified as a necessary step to protect against rampant cell death during cryopreservation. In addition to osmotic stress, cooling and thawing rates were also shown to have significant influence on cell survival during low temperature storage. In general, successful low-temperature cell preservation consists of the addition of a CPA (commonly 10% DMSO), alone or in combination with additional permeating or non-permeating agents, cooling rates of approximately 1ºC/min, and storage in either liquid or vapor phase nitrogen. In addition to general considerations, cell-specific recommendations for hepatocytes, pancreatic islets, sperm, oocytes, and stem cells should be observed to maximize yields. For example, rapid cooling is associated with better cryopreservation outcomes for oocytes, pancreatic islets, and embryonic stem cells while slow cooling is recommended for cryopreservation of hepatocytes, hematopoietic stem cells, and mesenchymal stem cells. Yields can be further maximized by implementing additional pre-cryo steps such as: pre-incubation with glucose and anti-oxidants, alginate encapsulation, and selecting cells within an optimal age range and functional ability. Finally, viability and functional assays are critical steps in determining the quality of the cells post-thaw and improving the efficiency of the current cryopreservation methods.

Islet transplantation has been shown to restore normoglycemia clinically. One of the current limitations to the widespread clinical use of islet transplantation is culturing and preserving more than 1 million islet equivalents in preparation for transplant. One possible solution is to bank frozen islets and use them when needed. Although promising, the standard islet freezing protocol introduces stress and cell death, resulting in high variability of islet quality post thawing. This study aimed to develop an improved cryopreservation protocol using alginate-encapsulated islets to improve islet survival and function for future transplants. Our data showed that encapsulation improved islet survival and function after thawing the frozen islets. Frozen encapsulated islets have an islet yield recovery of 84% when compared to non-encapsulated islets at 72% after thawing. Post-thaw viability was 78% for non-encapsulated islets compared to 88% for encapsulated islets. The stimulation index values after a static glucose test following thawing were 1.9 ± 0.5, 2.9 ± 0.1, and 3.3 ± 0.3 for the non-encapsulated, 1.75% alginate, and 2.5% alginate groups, respectively. In a transplant study, the mice that received 1.75% alginate-encapsulated cryopreserved islets achieved normoglycemia on average 5 days after transplant. In comparison, control mice that received fresh islets took 4 days, while those receiving unencapsulated cryopreserved islets took 18 days. In conclusion, encapsulating islets in 1.75% alginate prior to freezing was shown to improve islet survival, function post thawing, and graft response significantly when compared to islets frozen without encapsulation.

Transplantation of pancreatic islets within a biomaterial device is currently under investigation in clinical trials for the treatment of patients with type 1 diabetes (T1D). Patients’ preferences on such implants could guide the designs of next-generation implantable devices; however, such information is not currently available. We surveyed the preferences of 482 patients with T1D on the size, shape, visibility, and transplantation site of islet containing implants. More than 83% of participants were willing to receive autologous stem cells, and there was no significant association between implant fabricated by one’s own stem cell with gender (χ 2 (1, n = 468) = 0.28; P = 0.6) or with age (χ 2 (4, n = 468) = 2.92; P = 0.6). Preferred location for islet transplantation within devices was under the skin (52.7%). 48.3% preferred microscopic disks, and 32.3% preferred a thin device (like a credit card). Moreover, 58.4% preferred the implant to be as small as possible, 25.4% did not care about visibility, and 16.2% preferred their implants not to be visible. Among female participants, 81% cared about the implant visibility, whereas this number was 64% for male respondents (χ 2 test (1, n = 468) = 16.34; P < 0.0001). 22% of those younger than 50 years of age and 30% of those older than 50 did not care about the visibility of implant (χ 2 test (4, n = 468) = 23.69; P < 0.0001). These results suggest that subcutaneous sites and micron-sized devices are preferred choices among patients with T1D who participated in our survey.

For the advancement of porcine xenotransplantation for clinical use in type 1 diabetes mellitus, the concerns of a sustainable and safe digestion enzyme blend must be overcome. Incorporating good manufacturing practices (GMP) can facilitate this through utilizing GMP-grade enzymes. In conjunction, still taking into account the cost-effectiveness, a wide concern. We evaluated how GMP-grade enzyme blends impact our piglet islets and their long-term effects.  Preweaned porcine islets (PPIs) were isolated from 8- to 10-day-old pigs. Digestion enzyme blends, collagenase type V (Type V), collagenase AF-1 GMP-grade with collagenase NB 6 GMP-grade (AF-1 and NB 6), and collagenase AF-1 GMP-grade with collagenase neutral protease AF GMP-grade (AF-1 and NP AF) were compared. Islet quality control assessments, islet yield, viability, and function, were performed on days 3 and 7, and cell content was performed on day 7.  GMP-grade AF-1 and NB 6 (17,209 ± 2,730 islet equivalent per gram of pancreatic tissue [IE/g] on day 3, 9,001 ± 1,034 IE/g on day 7) and AF-1 and NP AF (17,214 ± 3,901 IE/g on day 3, 8,833 ± 2,398 IE/g on day 7) showed a significant increase in islet yield compared to Type V (4,618 ± 1,240 IE/g on day 3, 1,923 ± 704 IE/g on day 7). Islet size, viability, and function showed comparable results in all enzyme blends. There was no significant difference in islet cellular content between enzyme blends.  This study demonstrated a comparison of GMP-grade collagenase enzyme blends and a standard crude collagenase enzyme in preweaned-aged porcine, a novel topic in this age. GMP-grade enzyme blends of AF-1 and NB 6 and AF-1 and NP AF resulted in substantially higher yields and as effective PPIs compared to Type V. In the long run, considering costs, integrity, and sustainability, GMP-grade enzyme blends are more favorable for clinical application due to high reproducibility in comparison to undefined manufacturing processes of standard enzymes.

Five-Year Follow-Up After Clinical Islet Transplantation Edmond A. Ryan 1 , Breay W. Paty 1 , Peter A. Senior 1 , David Bigam 2 , Eman Alfadhli 1 , Norman M. Kneteman 2 , Jonathan R.T. Lakey 2 and A.M. James Shapiro 2 1 Department of Medicine, Clinical Islet Transplant Program, University of Alberta and Capital Health, Edmonton, Alberta, Canada 2 Department of Surgery, Clinical Islet Transplant Program, University of Alberta and Capital Health, Edmonton, Alberta, Canada Address correspondence and reprint requests to Edmond A. Ryan, Clinical Islet Transplant Program, 2000 College Plaza, 8215 112th St., Edmonton, Alberta, Canada T6G 2C8. E-mail: edmond.ryan{at}ualberta.ca Abstract Islet transplantation can restore endogenous β-cell function to subjects with type 1 diabetes. Sixty-five patients received an islet transplant in Edmonton as of 1 November 2004. Their mean age was 42.9 ± 1.2 years, their mean duration of diabetes was 27.1 ± 1.3 years, and 57% were women. The main indication was problematic hypoglycemia. Forty-four patients completed the islet transplant as defined by insulin independence, and three further patients received >16,000 islet equivalents (IE)/kg but remained on insulin and are deemed complete. Those who became insulin independent received a total of 799,912 ± 30,220 IE (11,910 ± 469 IE/kg). Five subjects became insulin independent after one transplant. Fifty-two patients had two transplants, and 11 subjects had three transplants. In the completed patients, 5-year follow-up reveals that the majority (∼80%) have C-peptide present post–islet transplant, but only a minority (∼10%) maintain insulin independence. The median duration of insulin independence was 15 months (interquartile range 6.2–25.5). The HbA 1c (A1C) level was well controlled in those off insulin (6.4% [6.1–6.7]) and in those back on insulin but C-peptide positive (6.7% [5.9–7.5]) and higher in those who lost all graft function (9.0% [6.7–9.3]) ( P < 0.05). Those who resumed insulin therapy did not appear more insulin resistant compared with those off insulin and required half their pretransplant daily dose of insulin but had a lower increment of C-peptide to a standard meal challenge (0.44 ± 0.06 vs. 0.76 ± 0.06 nmol/l, P < 0.001). The Hypoglycemic score and lability index both improved significantly posttransplant. In the 128 procedures performed, bleeding occurred in 15 and branch portal vein thrombosis in 5 subjects. Complications of immunosuppressive therapy included mouth ulcers, diarrhea, anemia, and ovarian cysts. Of the 47 completed patients, 4 required retinal laser photocoagulation or vitrectomy and 5 patients with microalbuminuria developed macroproteinuria. The need for multiple antihypertensive medications increased from 6% pretransplant to 42% posttransplant, while the use of statin therapy increased from 23 to 83% posttransplant. There was no change in the neurothesiometer scores pre- versus posttransplant. In conclusion, islet transplantation can relieve glucose instability and problems with hypoglycemia. C-peptide secretion was maintained in the majority of subjects for up to 5 years, although most reverted to using some insulin. The results, though promising, still point to the need for further progress in the availability of transplantable islets, improving islet engraftment, preserving islet function, and reducing toxic immunosuppression. CBC, complete blood count CMV, cytomegalovirus HOMA, homeostasis model assessment HYPO score, Hypoglycemic score LI, lability index LFT, liver function test PRA, panel reactive antibody Footnotes Accepted April 13, 2005. Received February 18, 2005. DIABETES

Islet transplantation is an evolving therapy that may be considered for patients with type 1 diabetes mellitus complicated by severe hypoglycemia or labile diabetes, provided all other attempts to stabilize glycemic control have been exhausted. This multicenter trial confirms that islet transplantation using the Edmonton protocol can successfully restore long-term endogenous production of insulin and glycemic stability in such patients, but insulin independence is usually not sustainable. Islet transplantation using the Edmonton protocol can successfully restore long-term endogenous production of insulin and glycemic stability in such patients, but insulin independence is usually not sustainable. Despite substantial improvements in insulin therapy and the care of patients with type 1 diabetes mellitus, a subgroup of patients is disabled by refractory hypoglycemia. Cell-based therapy with islet transplantation offers the possibility of improved glycemic control. The past three decades have witnessed substantial progress in islet transplantation. 1 – 3 Before the year 2000, few centers performing islet transplantation achieved high rates of sustainable insulin independence after this procedure among patients with type 1 diabetes mellitus. 1 – 3 In 2000, Shapiro et al. 4 reported their initial findings with up to a year of follow-up in seven consecutive subjects treated with glucocorticoid-free immunosuppressive . . .

Pre-weaned porcine islets (PPIs) represent an unlimited source for islet transplantation but are functionally immature. We previously showed that necrostatin-1 (Nec-1) immediately after islet isolation enhanced the in vitro development of PPIs. Here, we examined the impact of Nec-1 on the in vivo function of PPIs after transplantation in diabetic mice. PPIs were isolated from pancreata of 8–15-day-old, pre-weaned pigs and cultured in media alone, or supplemented with Nec-1 (100 µM) on day 0 or on day 3 of culture (n = 5 for each group). On day 7, islet recovery, viability, oxygen consumption rate, insulin content, cellular composition, insulin secretion capacity, and transplant outcomes were evaluated. While islet viability and oxygen consumption rate remained high throughout 7-day tissue culture, Nec-1 supplementation on day 3 significantly improved islet recovery, insulin content, endocrine composition, GLUT2 expression, differentiation potential, proliferation capacity of endocrine cells, and insulin secretion. Adding Nec-1 on day 3 of tissue culture enhanced the islet recovery, proportion of delta cells, beta-cell differentiation and proliferation, and stimulation index. In vivo, this leads to shorter times to normoglycemia, better glycemic control, and higher circulating insulin. Our findings identify the novel time-dependent effects of Nec-1 supplementation on porcine islet quantity and quality prior to transplantation.

Medical devices for cell therapy can be improved through prevascularization. In this work we study the vascularization of a porous polymer device, previously used by our group for pancreatic islet transplantation with results indicating improved glycemic control. Oxygen partial pressure within such devices was monitored non-invasively using an optical technique. Oxygen-sensitive tubes were fabricated and placed inside devices prior to subcutaneous implantation in nude mice. We tested the hypothesis that vascularization will be enhanced by administration of the pro-angiogenic factor hydrogen sulfide (H2S). We found that oxygen dynamics were unique to each implant and that the administration of H2S does not result in significant changes in perfusion of the devices as compared with control. These observations suggest that vascular perfusion and density are not necessarily correlated, and that the rate of vascularization was not enhanced by the pro-angiogenic agent.

Islet transplantation has been investigated as a treatment for type 1 diabetes mellitus in selected patients with inadequate glucose control despite insulin therapy. However, the perennial hope that such an approach would result in long-term freedom from the need for exogenous insulin, with stabilization of the secondary complications of diabetes, has failed to materialize in practice. Of the 267 allografts transplanted since 1990, only 12.4 percent have resulted in insulin independence for periods of more than one week, and only 8.2 percent have done so for periods of more than one year. 1 In the majority of these procedures, the regimen . . .

Attention deficit hyperactivity disorder (ADHD) is a chronic heritable developmental delay psychiatric disorder requiring chronic management, characterized by inattention, hyperactivity, hyperkinectivity and impulsivity. Subjective clinical evaluation still remains crucial in its diagnosis. Discussed are two key aspects in the \"characterizing ADHD\" and on the quest for objective \"pathognomonic/endophenotypic diagnostic markers of ADHD\". The first aspect briefly revolves around issues related to identification of pathognomonic/endophenotypic diagnostic markers in ADHD. Issues discussed include changes in ADHD definition, remission/persistence and overlapping-symptoms cum shared-heritability with its co-morbid cross-border mental disorders. The second aspect discussed is neurobiological and EEG-based studies on ADHD. Given the neurobiological and temporal aspects of ADHD symptoms the electroencephalograph (EEG) like NeuralScan by Medeia appears as an appropriate tool. The EEGs appropriateness is further enhanced when coupled with suitable behavior/cognitive/motor/psychological tasks/paradigms yielding EEG-based markers like event-related-potential (ERPs like P3 amplitudes and latency), reaction time variability (RTV), Theta: Beta ratio (TBR) and sensorimotor rhythm (SMR). At present, these markers could potentially help in the neurobiological characterization of ADHD and either help in identifying or lay the groundwork for identifying pathognomonic and/or endophenotypic EEG-based markers enabling its diagnosis, treatment and management. Keywords: ADHD, EEG, event related potential, reaction time