Vitrified Wharton’s jelly tissue as a biomaterial for multiple tissue engineering applications

Abstract

Wharton’s Jelly (WJ) tissue is a promising biomaterial, for tissue engineering applications. However, its preservation over a long period in order to be readily available needs further optimization. A possible solution could be the vitrification and storage of WJ tissue at low temperatures. The aim of this study is to evaluate the effect of low temperature in the WJ tissue, which was stored at ?196?°C, either with the vitrification or conventional cryopreservation methods. WJ tissues were isolated from human umbilical cords, cryopreserved with the above methods and remained for 1?year at ?196?°C. Histological analysis of tissue’s extracellular matrix (ECM), isolation, and characterization of mesenchymal stromal cells (MSCs) were performed. Histological analysis revealed the presence of ECM components such as collagen, sulfated glycosaminoglycans (sGAGs), and the presence of cell nuclei only in vitrified samples. Furthermore, MSCs were isolated and expanded successfully from vitrified WJ tissues, whereas a few viable cells were obtained from conventionally cryopreserved tissues that were not further expanded. In conclusion, this study indicated the proper preservation of vitrified WJ tissues after 1?year of storage, which eventually could be used in tissue engineering and regenerative medicine approaches.     

Anti-thymocyte globulin and post-transplant cyclophosphamide predisposes to inferior outcome when using cryopreserved stem cell grafts

During 2020, the concurrent novel COVID-19 pandemic lead to widespread cryopreservation of allogeneic hematopoietic cell transplant grafts based on National Marrow Donor Program and European Society of Blood and Marrow Transplantation recommendations, in order to secure grafts before the start of conditioning chemotherapy. We sought to examine the impact of this change in practice on patient outcomes. We analyzed the outcomes of 483 patients who received hematopoietic stem cell transplantation (HSCT) between August 2017 and August 2020, at Princess Margaret Cancer Centre, Canada, in the retrospective study, comparing the outcomes between those who received cryopreserved or fresh peripheral blood stem cell grafts. Overall compared with those who received fresh grafts (n = 348), patients who received cryopreserved grafts (n = 135) had reduced survival and GRFS, reduced incidence of chronic graft-versus-host disease (GvHD), delay in neutrophil engraftment, and higher graft failure (GF), with no significant difference in relapse incidence or acute GvHD. However, recipients of cryopreserved matched-related donor HSCT showed significantly worse OS, NRM, GRFS compared with fresh grafts. Multivariable analysis of the entire cohort showed significant impact of cryopreservation on OS, relapse, cGvHD, GF, and GRFS. We conclude that cryopreservation was associated with inferior outcomes post-HSCT, possibly due to the combination of ATG and post-transplant cyclophosphamide impacting differential tolerance to cryopreservation on components of the stem cell graft; further studies are warranted to elucidate mechanisms for this observation.     

Antioxidative effects of cysteamine,hyaluronan and fetuin on post‐thaw semen quality,DNA integrity and oxidative stress parameters in the Brown Swiss bull

The aim of this study was to compare the effectiveness of antioxidants including cysteamine (2.5, 7.5 mm ), hyaluronan (0.25, 1 mg ml^?1) and fetuin (5, 10 mg ml^?1) in the freezing of Brown Swiss bull semen. The best percentages of CASA motilities were achieved with 10 mg ml^?1 of fetuin and 2.5 mm of cysteamine. For sperm morphology, 10 mg ml^?1 of fetuin and 2.5 mm of cysteamine had better protective effects (P < 0.001). The results of hypo‐osmotic swelling test showed that the percentage values of membrane integrity in all the groups, excluding that supplemented with 5 mg ml^?1 of fetuin, were higher than those of the control group (P < 0.001). Results obtained for the DNA damage of sperm cells demonstrated that 0.25 mg ml^?1 of hyaluronan, and 2.5 and 7.5 mm of cysteamine led to lower rates of spermatozoa with damaged DNA, compared with the control group (P < 0.001). The maintenance of superoxide dismutase and glutathione peroxidase antioxidant activities following freeze‐thawing with 2.5 and 7.5 mm of cysteamine and 10 mg ml^?1 of fetuin was demonstrated to be at a higher level in comparison with the control group (P < 0.001). Malondialdehyde formation was found to be lower in the groups supplemented with 0.25 mg ml^?1 of hyaluronan and 7.5 mm of cysteamine after the freeze‐thawing process (P < 0.001).     

番茄红素对人精子冷冻损伤的保护作用及机制研究

目的:通过在人精子冷冻保护液中添加不同浓度的番茄红素,研究番茄红素对精子冷冻损伤的可能保护作用及机制。方法:选择吉林省人类精子库捐精者的精液标本25份,每份精液一式4份,3∶1加入冷冻保护液后混匀,不含番茄红素者设为对照组(Ly0),而Ly2、Ly5、Ly10实验组混合液中分别含有2、5、10μmol/L的番茄红素,冷冻复苏精液进行常规分析,采用流式细胞术分析冻融后各组精子的凋亡率;采用硫代巴比妥酸(TBA)比色法检测精子中脂质过氧化产物丙二醛(MDA)浓度。利用JC-1标记法检测线粒体膜电位。结果:冻融后各组精子运动参数较新鲜精液参数均明显下降(P<0.01),Ly5组冻融后精子凋亡率[(25.68±4.36)%]较对照组[(33.26±4.78)%]显著下降(P<0.05);Ly5组线粒体膜电位水平[(66.18±14.23)%]显著高于对照组[(55.24±12.31)%],P<0.05;Ly2、Ly5、Ly10组的MDA水平与对照组比较,无统计学差异(P>0.05)。结论:在精液冷冻保护液中添加一定浓度的番茄红素可以减少线粒体氧化损伤,减轻活性氧对精子质膜的氧化应激损伤,从而提高精子的抗凋亡能力。     

液氮冻存降低成骨细胞免疫原性的实验研究

目的:探讨液氮冷冻(fluid nitrogen cryopreservation)对成骨细胞免疫原性的影响。方法:体外培养成骨细胞,传代鉴定后液氮冻存3个月,复苏。利用流式细胞仪(flow cytometry,FCM),通过间接免疫荧光技术测定冻存前后成骨细胞的免疫原性。同时采用成骨细胞、淋巴细胞混合培养(osteoblasts and lymphocytes mixed culture,OLMC)的方法,测定液氮冷冻对成骨细胞免疫原性的影响。结果:体外培养细胞经鉴定为成骨细胞,液氮冻存3个月后存活率>90%。FCM及OLMC实验结果均表明冻存后成骨细胞的免疫原性显著降低(P<0.01)。结论:低温液氮冷冻是一种理想的降低成骨细胞免疫原性的方法。     

The Uruguayan semen donor population: A twenty-eight-year retrospective study

Several studies have reported a global decline in seminal quality over the years. The objective of this study was to describe the semen donor population of Uruguay through comparing data of successive samples banked by the same donors and the analysis of their semen and physical characteristics, ancestry origin and educational level. A total of 3,449 ejaculated samples collected from 71 donors, cryobanked between 1989 and March 2017 at Fertilab, were analysed. Results revealed a mean age of 23.90 ± 3.98 years, an average weight of 74.95 ± 1.09 kg and a mean height of 1.78 ± 0.06 m. The majority of the donors trace their origin to Europe (74.65%, 53/71) and 66.19% (47/71) have a level of education higher than secondary school. We observed longitudinal differences in two parameters, that is sperm concentration and semen volume. Sperm concentration declined, while semen volume increased significantly over the 28-year period. The results of the present study are in accordance with that of previous articles that also reported a decline in sperm concentration over time. However, no differences were observed in total sperm number per ejaculate due to the increase in semen volume values, thus reflecting no real changes in sperm production over time.     

High levels of oxidation–reduction potential in frozen-thawed human semen are significantly correlated with poor post-thaw sperm quality

This study examined the relationship between oxidation–reduction potential (ORP) in frozen-thawed semen and the post-thaw sperm parameters. Levels of ORP were measured in 25 samples from men presenting for routine infertility work-up and were expressed as millivolt (mV)/10⁶ sperm/ml. Frozen-thawed samples were examined for post-thaw total motility (TM%), progressive motility (PM%), total sperm count (TSC) and ORP. The cryo-survival rate (CSR) was calculated as post-thaw TM/pre-freeze TM × 100. Data are provided as median and interquartile range (25th and 75th percentiles). The post-thaw TM% (10.0% [4.00%, 15.1%]), PM% (5.88% [2.97%, 9.33%]) and TSC (12.5 [10.0, 17.5] × 10⁶ sperm) were significantly lower than the pre-freeze TM% (45.9% [32.9%, 59.1%], PM% (31.5% [24.4%, 40.0%] and TSC (120 [90, 250] ×10⁶ sperm) (p < .001). Post-thaw ORP (2.62 [2.52, 3.13] mV/10⁶ sperm/ml) was significantly higher than pre-freeze ORP (0.73 [0.54, 1.21] mV/10⁶ sperm/ml; p < .001). The CSR was 21.7% (11.3%, 31.9%). The post-thaw seminal ORP was negatively correlated with post-thaw TM% (r = −.5; p = .02), PM% (r = −.41; p = .03), TSC (r = −.60; p = .03) and CSR (r = −.52; p = .01). Increased levels of ORP are significantly correlated with poor post-thaw sperm quality and CSR.