Three-dimensional co-culture of human spermatogonial stem cells with Sertoli cells in soft agar culture system supplemented by growth factors and Laminin.

Application of a three-dimensional (3D) culture system for in vitro proliferation and differentiation of human spermatogonial stem cells (SSCs) is a useful tool for the investigation of the spermatogenesis process and the management of male infertility particularly in prepubertal cancer patients. The main purpose of this study was to investigate the proliferation of human SSCs co-cultured with Sertoli cells in soft agar culture system (SACS) supplemented by Laminin and growth factors. Testicular cells were isolated from testes of brain-dead patients and cultured in two-dimensional (2D) culture system for 3 weeks. After 3 weeks, functional SSCs were evaluated by xenotransplantation and also identification of cells was assessed by immunocytochemistry, flow cytometry, and RT-PCR. Then, SSCs and Sertoli cells were transferred to the upper layer of SACS for 3 weeks. After 3 weeks, the number of colonies and the expression of specific SSCs and Sertoli cell markers, as well as apoptotic genes were evaluated. Our results showed that transplanted SSCs, migrated into the basement membrane of seminiferous tubules of recipient mice. The expression of PLZF, α6-Integrin, and Vimentin proteins in SSCs and Sertoli cells were observed in 2D and 3D culture systems. The expression rate of PLZF, α6-Integrin, Bcl2, and colony number in SACS supplemented by Laminin and growth factors group were significantly higher than non-supplemented groups (P ≤ 0.01), but the expression rate of c-kit and Bax in supplemented group were significantly lower than non-supplemented groups (P ≤ 0.05). This 3D co-culture system decreased apoptosis and increased propagation of human SSCs. Therefore, this designed system can be utilized to increase the proliferation of human SSCs in prepubertal male cancer and azoospermic men to obtain an adequate SSCs number to outotransplant success and in vitro spermatogenesis.

Consequences of More Coordinator Engagement to Take Consent for Organ Donation: Comparisons of New Versus Experienced Staff.

OBJECTIVES: The method of obtaining family consent for organ donation after occurrence of brain death in Iran is an opt-in process. Because of complicated cultural, legal, religious, and familial structures in Iran, it is not simple to take consent for organ donation in brain death situations. The process needs the professional staff to be experienced and have an appropriate personality to obtain consent. MATERIALS AND METHODS: We previous used a limited number of experienced coordinators to take consent. We revised our decision 2 years ago because of lack of trained personnel. From the beginning of 2017, we have started training courses regarding approaching families for donations for all interested coordinators. After participants finished the theoretical and practical training, we allowed them to interview themselves to practice a first evaluation for a suitable potential donor. We have continued this strategy and have gathered feedback from participating coordinators. RESULTS: At the beginning of 2017, 9 coordinators who were previously qualified for handling other steps of organ donation were put into a compact training program to train for family interviews. In 2016, the experienced interviewers had a 88% success rate compared with a success rate of 83% in 2017 for inexperienced interviewers (P = .1). No significant differences were shown. Despite younger ages of donors obtained in 2017, median donated organs per donor decreased from 2.81 to 2.66. The average time interval between first contact with families and establishment of family consent was not significantly different between experienced and inexperienced coordinators (9.2 vs 8.7 h). CONCLUSIONS: Although with the new strategy we experiensed a decrease in the consent rate, we believe this strategy will have long-term beneficial effects for our organization and the decreased rate will be a temporary decline. Interviews showed that this strategy made our coordinators more motivated and responsible for their job. As a result, both volume and quality of organ procurement activities will be increased.

Liver Donation From Marginal Donors: To Donate or Not to Donate?

OBJECTIVES: Livers from deceased donors compose nearly 30% of all organ transplants, and about 700 liver transplants are carried out per year. Marginal livers (extended-criteria donors), however, are not usually accepted by recipient teams, and there is only one center for these procedures in Iran. The final decision is made according to criteria that are much more conservative than other globally accepted ones. MATERIALS AND METHODS: To find significant differences and construct an algorithm to predict the future of marginal livers (before transfer of donors to our organ procurement unit), successfully donated and unsuccessfully donated livers divided into 2 age groups were compared in terms of age, sex, body mass index, liver function tests, significant medical disorders, and preretrieval liver sonography report. RESULTS: In the first age group (50-59 years old), there were 88 successful donors and 27 unsuccessful cases (due to 3 reasons: positive virology tests, death before retrieval, and ruled out by surgeons in the operating room). In the second age group (? 60 years old), there were 45 successful donors and 11 unsuccessful cases. The results showed that there were no differences between successful and unsuccessful cases regarding age and liver sonography results; however, sex, body mass index, liver function tests, and previous medical disorder were different between groups. Donors in both age groups were mostly male (60% and 73%); however, surprisingly, most unsuccessful cases were female (56.6% and 57%). Donors had generally lower body mass index (2 numerical difference), better liver function tests, and less risk factors (heart disease, diabetes mellitus, fatty liver, and chronic alcohol consumption). CONCLUSIONS: Before transfer of marginal liver donors to the organ procurement unit for further evaluation and organ retrieval, one must consider that age is just a number and the presence of other comorbidities can have more decisive roles on liver quality.

Role of More Active Identification of Brain-Dead Cases in Increasing Organ Donation.

OBJECTIVES: Organ donor shortage is a worldwide problem, resulting in 10% to 30% mortality rates for patients on wait lists for organ transplant. For brain-dead patients in Iran, it is mandatory for intensive care unit patients with Glasgow Coma Scale below 5/15 to be reported to an organ procurement unit. However, this process has not been functioning effectively. Here, we present the effects of changing the strategies on detecting brain-dead cases on the organ donor pool. MATERIALS AND METHODS: From March 2015 to March 2016, we changed our strategy in active detection of brain-dead cases. Since March 2015, our newly established protocol for active detection of brain-dead cases includes the following changes: (1) instead of calling high-volume intensive care units 3 times per week, we switched to calling every day in the morning; (2) instead of calling low-volume intensive care units 1 time per week, we switched to calling 3 times per week; (3) we included intensive care units (cardiac and general), neurosurgery, and emergency departments, as well as nursing supervisor offices, in our call and visit lists; and (4) we increased visits to wards by our trained staff as inspectors. RESULTS: From March 2015 to March 2016, the number of reported suspected brain-dead cases has increased from 224 to 460 per year, with proven brain death increasing from 180 to 306 cases. The actual number of donors has also increased, from 116 to 165 donations (53% increase) over 1 year. CONCLUSIONS: More proactive strategies have had significant effects on brain-dead detection, resulting in significantly increased donor pools and organ donations. In countries with low cooperation of hospital staff, more proactive engagement in detecting brain-dead cases is a good solution to prevent loss of potential organ donors, with a final result of decreasing wait list mortality.

Impact of a Full-Time Donor Management Protocol on Donors' Liver Biopsy Findings: Progress to Date.

OBJECTIVES: This study investigated a fixed coordinator-directed donor management strategy's impact on donated liver quality, as determined by definitive biopsy results. MATERIALS AND METHODS: We collected donated liver biopsy results from donations both before and after implementing a fixed coordinator-directed donor management strategy. This strategy involved full-time attendance by a donor coordinator and continued resuscitation of brain-dead donors. All donations took place in a single organ procurement unit. We also followed up results of biopsies from the Liver Transplantation Center database of Namazi Hospital in Shiraz, Iran. RESULTS: We compared biopsy findings of 192 livers donated from 2012 to 2013 (group A) with 276 livers donated from 2015 until August 2016 (group B). Data analysis showed that 67 livers (34.9%) in group A were rejected for transplant owing to severe steatosis in 17 (8.9%), moderate/severe fibrosis in 9 (4.7%), moderate/severe necrosis in 28 (14.6%), and 13 (6.8%) rejected for other pathologies. Among group B livers, 59 (21.4%) were not deemed suitable for transplant owing to severe steatosis in 37 (13.5%), moderate/severe fibrosis in 6 (2.1%), and moderate/ severe necrosis in 16 (5.7%). Overall, steatosis was found in 94 livers (49.2%) in group A versus 175 livers (63.3%) in group B (P = .007). Donor age in group A averaged 36.5 years versus 47.9 years in group B (P = .02). Necrosis was found in 33 livers (17.2%) in group A and 22 livers (7.9%) in group B (P = .008). One-month survival rates were 95.3% and 96.3% for groups A and B (P = .08). CONCLUSIONS: Donated liver disqualification before transplant noticeably decreased despite the shift in demographic patterns from 2012 to 2016. In group A, brain-dead liver donors were younger and more often died from trauma, whereas group B donors had more cerebrovascular accident-induced deaths. This achievement took place alongside increased rates of steatosis and decreased rates of necrosis.