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Annals of Biomedical Engineering Mar 2023Liver cryopreservation has the potential to enable indefinite organ banking. This study investigated vitrification-the ice-free cryopreservation of livers in a...
Liver cryopreservation has the potential to enable indefinite organ banking. This study investigated vitrification-the ice-free cryopreservation of livers in a glass-like state-as a promising alternative to conventional cryopreservation, which uniformly fails due to damage from ice formation or cracking. Our unique "nanowarming" technology, which involves perfusing biospecimens with cryoprotective agents (CPAs) and silica-coated iron oxide nanoparticles (sIONPs) and then, after vitrification, exciting the nanoparticles via radiofrequency waves, enables rewarming of vitrified specimens fast enough to avoid ice formation and uniformly enough to prevent cracking from thermal stresses, thereby addressing the two main failures of conventional cryopreservation. This study demonstrates the ability to load rat livers with both CPA and sIONPs by vascular perfusion, cool them rapidly to an ice-free vitrified state, and rapidly and homogenously rewarm them. While there was some elevation of liver enzymes (Alanine Aminotransferase) and impaired indocyanine green (ICG) excretion, the nanowarmed livers were viable, maintained normal tissue architecture, had preserved vascular endothelium, and demonstrated hepatocyte and organ-level function, including production of bile and hepatocyte uptake of ICG during normothermic reperfusion. These findings suggest that cryopreservation of whole livers via vitrification and nanowarming has the potential to achieve organ banking for transplant and other biomedical applications.
Topics: Rats; Vitrification; Cryopreservation; Cryoprotective Agents; Hepatocytes; Liver; Animals
PubMed: 36183025
DOI: 10.1007/s10439-022-03064-2 -
Current Protocols in Microbiology Dec 2019Shigella species, which are closely related to Escherichia coli, can easily be maintained and stored in the laboratory. This article includes protocols for preparation...
Shigella species, which are closely related to Escherichia coli, can easily be maintained and stored in the laboratory. This article includes protocols for preparation of routine growth conditions and media, for storage of the bacteria, and for monitoring of the presence of the virulence plasmid. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Growth of S. flexneri from frozen stocks or agar stabs Basic Protocol 2: Growth of S. flexneri in rich liquid medium Alternate Protocol 1: Growth of S. flexneri in rich defined medium Alternate Protocol 2: Growth of S. flexneri in minimal medium Basic Protocol 3: Storage of S. flexneri in frozen stocks Alternate Protocol 3: Storage of S. flexneri in agar stabs.
Topics: Bacteriological Techniques; Culture Media; Plasmids; Preservation, Biological; Shigella
PubMed: 31816179
DOI: 10.1002/cpmc.93 -
BioMed Research International 2022Achai is a small size cattle breed, resilient to harsh and cold environment. Cryopreservation of Achai bull semen may help to improve its genetics and preserve the...
Achai is a small size cattle breed, resilient to harsh and cold environment. Cryopreservation of Achai bull semen may help to improve its genetics and preserve the germplasm. Reactive oxygen species (ROS) affects the structural and functional integrity of the spermatozoa. During freezing and thawing processes, the ROS make changes in the spermatozoa quality parameters and reduce total antioxidant capacity (T-AOC) of semen that is considered as marker of oxidative stress. This study was designed to determine the effect of glycine along with vitamin E on post-thawed spermatozoa quality and total antioxidant capacity in Achai cattle. The semen collection was done twice a week from four mature fertile Achai cattle bulls ( = 4). The glycine was utilized as 0 mM, 5 mM, 10 mM, 15 mM, and 20 mM along with vitamin E @ 2.3 mM added constantly in each concentration. The control group contained all extenders except glycine. The results revealed that post-thawed spermatozoa motility was found significantly higher ( < 0.05) at 10 mM as compared to 5 mM, 15 mM, and 20 mM. Compared with control group, glycine concentration at 10 mM and other concentrations increased progressive and fast motility (%), curvilinear, straight line, and average path velocity (m/s). Moreover, beat cross frequency (Hz) was higher ( < 0.05), and post-thaw viability (%), plasma membrane integrity, and mitochondrial membrane potential were significantly higher ( < 0.05) at 10 mM of glycine concentration in comparison to control and other glycine concentrations. Besides, acrosome integrity (%) and DNA integrity (%) as well as post-thawed T-AOC were also significantly higher ( < 0.05) at 10 mM of glycine concentration as compared to other glycine concentrations and control group. It is concluded that 10 mM of glycine along with vitamin E @ 2.3 mM improved cryopreserved semen quality of Achai bull.
Topics: Animals; Antioxidants; Cattle; Cryopreservation; Fabaceae; Glycine; Male; Plant Breeding; Reactive Oxygen Species; Semen Analysis; Semen Preservation; Spermatozoa; Vitamin E
PubMed: 35968237
DOI: 10.1155/2022/8282387 -
Nature Communications Oct 2022Deoxyribonucleic acid (DNA) is the blueprint of life, and cost-effective methods for its long-term storage could have many potential benefits to society. Here we present...
Deoxyribonucleic acid (DNA) is the blueprint of life, and cost-effective methods for its long-term storage could have many potential benefits to society. Here we present the method of in situ cryosilicification of whole blood cells, which allows long-term preservation of DNA. Importantly, our straightforward approach is inexpensive, reliable, and yields cryosilicified samples that fulfill the essential criteria for safe, long-term DNA preservation, namely robustness against external stressors, such as radical oxygen species or ultraviolet radiation, and long-term stability in humid conditions at elevated temperatures. Our approach could enable the room temperature storage of genomic information in book-size format for more than one thousand years (thermally equivalent), costing only 0.5 $/person. Additionally, our demonstration of 3D-printed DNA banking artefacts, could potentially allow 'artificial fossilization'.
Topics: Humans; Ultraviolet Rays; DNA; Blood Preservation; Preservation, Biological; Oxygen
PubMed: 36270991
DOI: 10.1038/s41467-022-33759-y -
PloS One 2019Dextranol, a reduced dextran, prevents damage to stored dry protein samples that unmodified dextran would otherwise cause. Desiccation protectants (xeroprotectants) like...
Dextranol, a reduced dextran, prevents damage to stored dry protein samples that unmodified dextran would otherwise cause. Desiccation protectants (xeroprotectants) like the polysaccharide dextran are critical for preserving dried protein samples by forming a rigid glass that protects entrapped protein molecules. Stably dried proteins are important for maintaining critical information in clinical samples like blood serum as well as maintaining activity of biologic drug compounds. However, we found that dextran reacts with both dried serum proteins and lyophilized purified proteins during storage, producing high-molecular weight Amadori-product conjugates. These conjugates appeared in a matter of days or weeks when stored at elevated temperatures (37° or 45°C), but also appeared on a timescale of months when stored at room temperature. We synthesized a less reactive dextranol by reducing dextran's anomeric carbon from an aldehyde to an alcohol. Serum samples dried in a dextranol-based matrix protected the serum proteins from forming high-molecular weight conjugates. The levels of four cancer-related serum biomarkers (prostate specific antigen, neuropilin-1, osteopontin, and matrix-metalloproteinase 7) decreased, as measured by immunoassay, when serum samples were stored for one to two weeks in dextran-based matrix. Switching to a dextranol-based xeroprotection matrix slightly reduced the damage to osteopontin and completely stopped any detectable damage during storage in the other three biomarkers when stored for a period of two weeks at 45°C. We also found that switching from dextran to dextranol in a lyophilization formulation eliminates this unwanted reaction, even at elevated temperatures. Dextranol offers a small and easy modification to dextran that significantly improves the molecule's function as a xeroprotectant by eliminating the potential for damaging protein-polysaccharide conjugation.
Topics: Blood Proteins; Desiccation; Dextrans; Drug Compounding; Oxidation-Reduction; Preservation, Biological; Protein Stability; Temperature
PubMed: 31490977
DOI: 10.1371/journal.pone.0222006 -
The Journal of Heart and Lung... Jun 2023Heart transplantation in donation after circulatory death (DCD) relies on warm perfusion using either in situ normothermic regional perfusion (NRP) or ex situ...
Ex-situ oxygenated hypothermic machine perfusion in donation after circulatory death heart transplantation following either direct procurement or in-situ normothermic regional perfusion.
BACKGROUND
Heart transplantation in donation after circulatory death (DCD) relies on warm perfusion using either in situ normothermic regional perfusion (NRP) or ex situ normothermic machine perfusion. In this study, we explore an alternative: oxygenated hypothermic machine perfusion (HMP) using a novel clinically applicable perfusion system, which is compared to NRP with static cold storage (SCS).
METHODS
In a porcine model, a DCD setting was simulated, followed by either (1) NRP and SCS (2) NRP and HMP with the XVIVO Heart preservation system or (3) direct procurement (DPP) and HMP. After preservation, heart transplantation (HTX) was performed. After weaning from cardiopulmonary bypass (CPB), biventricular function was assessed by admittance and Swan-Ganz catheters.
RESULTS
Only transplanted hearts in the HMP groups showed significantly increased biventricular contractility (end-systole elastance) 2 hour post-CPB (left ventricle absolute change: NRP HMP: +1.8 ± 0.56, p = 0.047, DPP HMP: +1.5 ± 0.43, p = 0.045 and NRP SCS: +0.97 ± 0.47 mmHg/ml, p = 0.21; right ventricle absolute change: NRP HMP: +0.50 ± 0.12, p = 0.025, DPP HMP: +0.82 ± 0.23, p = 0.039 and NRP SCS: +0.28 ± 0.26, p = 0.52) while receiving significantly less dobutamine to maintain a cardiac output >4l/min compared to SCS. Diastolic function was preserved in all groups. Post-HTX, both HMP groups showed significantly less increments in plasma troponin T compared to SCS.
CONCLUSION
In DCD HTX, increased biventricular contractility post-HTX was only observed in hearts preserved with HMP. In addition, the need for inotropic support and signs of myocardial damage were lower in the HMP groups. DCD HTX can be successfully performed using DPP followed by preservation with HMP in a preclinical setting.
Topics: Swine; Animals; Humans; Organ Preservation; Perfusion; Heart Transplantation; Extracorporeal Circulation; Heart; Tissue Donors; Death; Tissue and Organ Procurement
PubMed: 36918339
DOI: 10.1016/j.healun.2023.01.014 -
BMJ Open Ophthalmology Nov 2022In the past decades, the human amniotic membrane has been largely applied for several surgical and non-surgical procedures. It has been farther demonstrated that both...
PURPOSE
In the past decades, the human amniotic membrane has been largely applied for several surgical and non-surgical procedures. It has been farther demonstrated that both hAM and cornea share similar patterns of expression of structural components of the basement membrane (like laminin 5 and collagen IV) making hAM an useful tissue for ocular surface reconstruction. Since 1996 in fact, amniotic membrane transplantation has been applied to a large number of ocular surface diseases including Stevens-Johnson syndrome, pterygium, corneal ulceration, ocular surface reconstruction after chemical/thermal burns and in the reconstruction after excision of ocular surface neoplasia. During the previous decades, hAM has achieved a pivotal role in regenerative medicine too.The possibility to preserve human amniotic membrane, without affecting membrane's features, has become pivotal, allowing virological and microbiological analyses to be carried out before grafting. The purpose of the present study is to investigate an easier and cheaper protocol for human amniotic membrane preservation without affecting its properties and structure, ensuring the safety profile of the tissue. We compared the effects on adhesive and structural properties of newer preservation conditions to those obtained with an established, standardized protocol (dimethyl sulfoxide at -160°C). In attempt to simplify and enhance the safety of the procedure, we tested dextran-based freezing medium and a dry condition (no medium) at temperatures of -80°C.
METHODS
Five patches of human amniotic membrane were obtained from three different donors. For each donor, five preservation condition were tested: dimethyl sulfoxide at -160°C, dimethyl sulfoxide at -80°C, dextran-based medium at -160°C, dextran-based medium at -80°C and dry freezing at -80°C (no medium). At the end of four months storage period, adhesive properties and structure were analyzed.
RESULTS
None of the newer preservation protocols showed differences in adhesive and structural properties of the tissues. The stromal layer always kept its adhesiveness, while both structure and basement membrane were not altered by any the preservation protocol.
CONCLUSIONS
Switching from liquid nitrogen cryopreservation to -80°C would reduce manipulation, simplify the procedure, making it also cheaper. The use of dextran-based freezing medium or no medium at all (dry condition) would avoid the potential toxicity of the dimethyl sulfoxide-based freezing media.
Topics: Humans; Amnion; Dimethyl Sulfoxide; Dextrans; Cornea; Cryopreservation
PubMed: 37282679
DOI: 10.1136/bmjophth-2022-EEBA.23 -
American Journal of Transplantation :... Apr 2021Ex situ normothermic machine perfusion (NMP) is being used increasingly in the assessment of higher risk deceased donor organs and to facilitate prolonged organ storage.... (Review)
Review
Ex situ normothermic machine perfusion (NMP) is being used increasingly in the assessment of higher risk deceased donor organs and to facilitate prolonged organ storage. Third-party packed red blood cells (pRBCs) are often used as an oxygen carrier in the perfusate of ex situ NMP. Despite the increasing interest in NMP, comparatively little attention has been paid to the appropriate selection of pRBCs. This includes the choice of ABO blood group and Rhesus D status, the need for special requirements for selected recipients, and the necessity for traceability of blood components. Flushing organs with cold preservation solution after NMP removes the overwhelming majority of third-party allogeneic pRBCs, but residual pRBCs within the organ may have biologically relevant effects following implantation as they enter the recipient's circulation. This review considers these issues, and suggests that national transplant and blood transfusion agencies work together to develop a co-ordinated approach within each country. This is especially important given the possibility of organ re-allocation between centers after ex situ NMP, and the ongoing development of organ perfusion hubs.
Topics: Cold Ischemia; Erythrocytes; Liver; Liver Transplantation; Organ Preservation; Perfusion
PubMed: 33048419
DOI: 10.1111/ajt.16355 -
International Journal of Molecular... Jan 2021Sex reversal has been used as a breeding strategy by salmonid fish to produce genetically and phenotypically single sex populations. Production of all-female fish has... (Review)
Review
Sex reversal has been used as a breeding strategy by salmonid fish to produce genetically and phenotypically single sex populations. Production of all-female fish has great importance for the creation of monosex female triploids of salmonid fish, which are valued for their sterility, lack of female maturation, and larger commercial size. Among salmonids, the majority of rainbow trout () production is based on all-female production with a high proportion of all-female triploid production in Europe. The main aim of this review is to present the recent knowledge regarding sex-reversed females (SRFs) of salmonid fish. We discuss the methods of sex reversal as well as their effects on the morphology and histology of the reproductive tract. We focus on the characteristics of SRF semen as well as the factors determining semen quality. The lower quality of SRF sperm compared to that of normal males has resulted in the need for the artificial maturation of semen. Most importantly, methods of semen storage-both short-term and long-term (cryopreservation)-that can improve hatchery operations are presented with the special emphasis on recent progress in development of efficient cryopreservation procedures and use of cryopreserved semen in hatchery practice. Moreover, we also address the emerging knowledge concerning the proteomic investigations of salmonid sperm, focusing primarily on the proteomic comparison of normal male and SRF testicular semen and presenting changes in SRF rainbow trout sperm proteome after in vitro incubation in artificial seminal plasma.
Topics: Animals; Cryopreservation; Female; Male; Oncorhynchus mykiss; Semen; Semen Analysis; Semen Preservation; Sex Determination Processes
PubMed: 33478050
DOI: 10.3390/ijms22020964 -
Reproduction & Fertility Apr 2023Sperm cryopreservation is a valuable tool for the long-term preservation of male fertility. Thus, determining the optimal technique for isolating spermatozoa post-thaw...
ABSTRACT
Sperm cryopreservation is a valuable tool for the long-term preservation of male fertility. Thus, determining the optimal technique for isolating spermatozoa post-thaw is vital to ensure recovery of the highest quality spermatozoa with minimal iatrogenic damage. This not only enhances the chances of successful conception but also reduces the risk of genetic damage in the embryo. To address this issue, human semen samples were cryopreserved using a slow freezing protocol and Quinn's Advantage™ Sperm Freeze medium. The samples were subsequently thawed and subjected to three types of sperm isolation procedures: direct swim-up, density gradient centrifugation, and electrophoretic separation using the Felix™ device. Cryopreservation led to the anticipated loss of sperm motility and vitality in association with increases in lipid peroxidation and DNA damage. Following sperm selection, all three isolation techniques resulted in an increase in sperm motility which was particularly evident with the swim-up and Felix™ procedures. The latter also significantly improved sperm vitality. There were no differences between sperm separation techniques with respect to morphology, and mitochondrial reactive oxygen species generation remained essentially unchanged when cell vitality was taken into account. By contrast, major differences were observed in DNA integrity and lipid aldehyde formation, where Felix™ isolated cells exhibiting significantly less DNA damage than the other isolation procedures as well as lower levels of 4-hydroxynonenal formation. Electrophoretic sperm isolation, therefore, offers significant advantages over alternative separation strategies, in terms of the quality of the gametes isolated and the time taken to achieve the isolation.
LAY SUMMARY
Long-term storage of sperm is vital to assisted reproductive technology because it permits the preservation of fertility that might be compromised as a result of factors such as chemotherapy or vasectomy. This goal can be achieved via cryopreservation - the freezing of cells to -196°C. When the sperm are subsequently required for conception, they must be carefully separated from the cryopreservation medium in a manner that maximizes the chances of successful conception and minimizes the risk of genetic defects in the offspring. In this paper, three isolation techniques were compared for their ability to separate ideal sperm from semen and media following cryopreservation. It was found that cryopreservation led to lower levels of motility and vitality and created higher levels of DNA and cell membrane damage. Of the three techniques compared, only cells separated on the basis of their size and electric charge (electrophoretic isolation) exhibited significantly lower levels of DNA fragmentation.
Topics: Humans; Male; Animals; Sperm Motility; Semen Preservation; Spermatozoa; Cryopreservation; Semen
PubMed: 37000632
DOI: 10.1530/RAF-22-0133