Assessing Attitudes and Understanding After Ovarian Tissue Cryopreservation: A Follow-Up Telephone Interview Survey.

Purpose: Assessing patient and guardian experiences regarding their history of ovarian tissue cryopreservation (OTC) years after initial procedure. Methods: Cross-sectional follow-up telephone survey. A questionnaire developed by The Pediatric Initiative Network of the Oncofertility Consortium, modified to assess intent and attitudes regarding OTC, tissue access knowledge, financial burden of tissue storage, and intent to use tissue, was utilized. Interviews were conducted for those who underwent OTC at a metropolitan children's hospital between 2013 and 2022. Results: Of 60 eligible patients, 39 interviews were completed. Contacted patients were 3-28 years old, with minors accompanied by guardians. Average age at OTC was 8.5 years old, and 5.1% (2/39) were deceased at the time of contact. All interviewees underwent OTC for fertility preservation before gonadotoxic treatment. Seventy percent of patients (7/10) and 48.1% (13/27) of guardians stated they would use frozen tissue for pregnancy, with 50% (5/10) of patients and 59.3% (16/27) of guardians not understanding tissue access. Regret occurred in 10% (1/10) of patients and 3.4% (1/29) of guardians. It was associated with 10.8% (4/37) of tissue discard due to failed storage payments. Financial concerns occurred in 29.7% (11/37) of interviewees. Overall, 92.3% (36/39) would recommend OTC, and 94.9% (37/39) would repeat their choice to undergo OTC. Conclusion: Follow-up after OTC is essential to patient understanding of tissue status, access, and payments. Most do not regret OTC, except in cases of financial burden leading to tissue discard. Follow-up should be sequentially scheduled and include counseling on financial assistance programs.

Impact of cryopreservation on elastomuscular artery mechanics.

Low temperatures slow or halt undesired biological and chemical processes, protecting cells, tissues, and organs during storage. Cryopreservation techniques, including controlled media exchange and regulated freezing conditions, aim to mitigate the physical consequences of freezing. Dimethyl sulfoxide (DMSO), for example, is a penetrating cryoprotecting agent (CPA) that minimizes ice crystal growth by replacing intracellular water, while polyvinyl alcohol (PVA) is a nonpenetrating CPA that prevents recrystallization during thawing. Since proteins and ground substance dominate the passive properties of soft biological tissues, we studied how different freezing rates, storage temperatures, storage durations, and the presence of cryoprotecting agents (5% [v/v] DMSO + 1 mg/mL PVA) impact the histomechanical properties of the internal thoracic artery (ITA), a clinically relevant blood vessel with both elastic and muscular characteristics. Remarkably, biaxial mechanical analyses failed to reveal significant differences among the ten groups tested, suggesting that mechanical properties are virtually independent of the cryopreservation technique. Scanning electron microscopy revealed minor CPA-independent delamination in rapidly frozen samples, while cryoprotected ITAs had better post-thaw viability than their unprotected counterparts using methyl thiazole-tetrazolium (MTT) metabolic assays, especially when frozen at a controlled rate. These results can be used to inform ongoing and future studies in vascular engineering, physiology, and mechanics.

Histochemical and morphological evaluation of a glyoxal acid-free fixative.

The application of most chemical fixatives, such as formalin, in the anatomic pathology laboratory requires safety training and hazardous chemical monitoring due to the toxicity and health risks associated with their use. Consequently, the use of formalin has been banned in most applications in Europe; the primary exception is its use in the histology laboratory in lieu of a suitable and safer alternative. Glyoxal based solutions, several of which are available commercially, are the most promising alternative fixatives, because they are based on a mechanism of fixation similar to that of formalin. Unlike formalin, however, glyoxal based solutions do not dissociate from water and therefore do not require ventilation measures such as a fume hood. A primary barrier to the adoption of commercially available glyoxal based solutions is their low pH, which can produce undesirable morphological and antigenic tissue alterations; however, a recently available neutral pH glyoxal product (glyoxal acid free) (GAF) has been developed to mitigate the challenges of low pH. We compared the morphology and histochemistry among tissues fixed in 10% neutral buffered formalin, a commercially available acidic glyoxal product (Prefer), and GAF. Tissues fixed in formalin and Prefer exhibited similar morphology and staining properties; tissues fixed with 2% GAF exhibited deleterious effects.

Engineered Test Tissues: A Model for Quantifying the Effects of Cryopreservation Parameters.

Engineered tissues are showing promise as implants to repair or replace damaged tissues in vivo or as in vitro tools to discover new therapies. A major challenge of the tissue engineering field is the sample preservation and storage until their transport and desired use. To successfully cryopreserve tissue, its viability, structure, and function must be retained post-thaw. The outcome of cryopreservation is impacted by several parameters, including the cryopreserving agent (CPA) utilized, the cooling rate, and the storage temperature. Although a number of CPAs are commercially available for cell cryopreservation, there are few CPAs designed specifically for tissue cryostorage and recovery. In this study, we present a flexible, relatively high-throughput method that utilizes engineered tissue rings as test tissues for screening the commercially available CPAs and cryopreservation parameters. Engineered test tissues can be fabricated with low batch-to-batch variability and characteristic morphology due to their endogenous extracellular matrix, and they have mechanical properties and a ring format suitable for testing with standard methods. The tissues were grown for 7 days in standard 48-well plates and cryopreserved in standard cryovials. The method allowed for the quantification of metabolic recovery, tissue apoptosis/necrosis, morphology, and mechanical properties. In addition to establishing the method, we tested different CPA formulations, freezing rates, and freezing points. Our proposed method enables timely preliminary screening of CPA formulations and cryopreservation parameters that may improve the storage of engineered tissues.

Effect of storage media on chondrocyte viability during cold storage of osteochondral dowels.

Osteochondral allograft transplantation is a successfully proven method to repair articular cartilage defects and prevent the degenerative effects of osteoarthritis. The number of osteochondral transplantations that can be performed each year is limited by availability of donor cartilage tissue and storage time constraints. Osteochondral transplantation success has been linked to high chondrocyte viability of the donor cartilage tissue at the time of implantation. Determining optimal storage conditions for donor cartilage is essential for tissue banks to safely provide quality cartilage tissue. In this study, we compared three tissue/cell media (DMEM/F12, RPMI-1640 and X-VIVO 10) for their ability to maintain chondrocyte viability during hypothermic storage for 28 days. Porcine osteochondral dowels were stored in each media for 28 days and cell viability was assessed every 7 days. Over the 28 day storage period, the chondrocyte viability of dowels stored in DMEM/F12, RPMI-1640, and X-VIVO 10 media all declined in a similar fashion. Our results show that all three media were equivalent in their ability to maintain cell viability of the cartilage tissue and provides rationale for the use of lower cost cell media (DMEM/F12 and RPMI-1640) for hypothermic storage of articular cartilage tissue.

Preservation of Mechanical and Morphological Properties of Porcine Cardiac Outflow Vessels after Decellularization and Wet Storage.

Widely used storage methods, including freezing or chemical modification, preserve the sterility of biological tissues but degrade the mechanical properties of materials used to make heart valve prostheses. Therefore, wet storage remains the most optimal option for biomaterials. Three biocidal solutions (an antibiotic mixture, an octanediol-phenoxyethanol complex solution, and a glycerol-ethanol mixture) were studied for the storage of native and decellularized porcine aorta and pulmonary trunk. Subsequent mechanical testing and microstructural analysis showed a slight increase in the tensile strength of native and decellularized aorta in the longitudinal direction. Pulmonary trunk elongation increased 1.3-1.6 times in the longitudinal direction after decellularization only. The microstructures of the tested specimens showed no differences before and after wet storage. Thus, two months of wet storage of native and decellularized porcine aorta and pulmonary trunks does not significantly affect the strength and elastic properties of the material. The wet storage protocol using alcohol solutions of glycerol or octanediol-phenoxyethanol mixture may be intended for further fabrication of extracellular matrix for tissue-engineered biological heart valve prostheses.

Survival of rat sciatic nerve segments preserved in storage solutions ex vivo assessed by novel electrophysiological and morphological criteria.

Most organ or tissue allografts with viable cells are stored in solutions ex vivo for hours to several days. Most allografts then require rapid host revascularization upon transplantation to maintain donor-cell functions (e.g., cardiac muscle contractions, hepatic secretions). In contrast, peripheral nerve allografts stored ex vivo do not require revascularization to act as scaffolds to guide outgrowth by host axons at 1-2 mm/d, likely aided by viable donor Schwann cells. Using current storage solutions and protocols, axons in all these donor organ/tissue/nerve transplants are expected to rapidly become non-viable due to Wallerian degeneration within days. Therefore, ex vivo storage solutions have not been assessed for preserving normal axonal functions, i.e., conducting action potentials or maintaining myelin sheaths. We hypothesized that most or all organ storage solutions would maintain axonal viability. We examined several common organ/tissue storage solutions (University of Wisconsin Cold Storage Solution, Normosol-R, Normal Saline, and Lactated Ringers) for axonal viability in rat sciatic nerves ex vivo as assessed by maintaining: (1) conduction of artificially-induced compound action potentials; and (2) axonal and myelin morphology in a novel assay method. The ten different storage solution conditions for peripheral nerves with viable axons (PNVAs) differed in their solution composition, osmolarity (250-318 mOsm), temperature (4°C vs. 25°C), and presence of calcium. Compound action potentials and axonal morphology in PNVAs were best maintained for up to 9 days ex vivo in calcium-free hypotonic diluted (250 mOsm) Normosol-R (dNR) at 4°C. Surprisingly, compound action potentials were maintained for only 1-2 days in UW and NS at 4°C, a much shorter duration than PNVAs maintained in 4°C dNR (9 days) or even in 25°C dNR (5 days). Viable axons in peripheral nerve allografts are critical for successful polyethylene glycol (PEG)-fusion of viable proximal and distal ends of host axons with viable donor axons to repair segmental-loss peripheral nerve injuries. PEG-fusion repair using PNVAs prevents Wallerian degeneration of many axons within and distal to the graft and results in excellent recovery of sensory/motor functions and voluntary behaviors within weeks. Such PEG-fused PNVAs, unlike all other types of conventional donor transplants, are immune-tolerated without tissue matching or immune suppression. Preserving axonal viability in stored PNVAs would enable the establishment of PNVA tissue banks to address the current shortage of transplantable nerve grafts and the use of stored PEG-fused PNVAs to repair segmental-loss peripheral nerve injuries. Furthermore, PNVA storage solutions may enable the optimization of ex vivo storage solutions to maintain axons in other types of organ/tissue transplants.

Polyploidy increases storage but decreases structural stability in Arabidopsis thaliana.

Whole-genome duplication, leading to polyploidy and endopolyploidy, is widespread throughout the tree of life.1-3 Both polyploidy and endopolyploidy can increase cell size via nucleotypic effects, but the phenotypic consequences of increased cell size at the tissue and whole-organism levels are less well understood.1-4 We quantified the consequences of autopolyploidy and endopolyploidy in nine diploid accessions of Arabidopsis thaliana, representing a gradient in endopolyploidy, to their corresponding experimentally synthesized neo-tetraploid and neo-octoploid cytotypes. The increase in cell size following genome duplication increased plant storage capacity, which increased tolerance of resource limitation, but also incurred biomechanical costs because of a reduction in the amount of cell wall per unit tissue volume. Our findings also show that the functional consequences of autopolyploidy can vary with accession identity, and the presence of this variation suggests that there is potential for adaptation following whole-genome duplication.

Optimisation of processing methods to improve success in the derivation of human multipotent mesenchymal stromal cells from cryopreserved umbilical cord tissue fragments.

Wharton's Jelly (WJ)-derived Mesenchymal Stromal Cells (MSC) are currently in the spotlight for the development of innovative MSC-based therapies due to their ease of sourcing, high proliferation capacity and improved immunopotency over MSC from other tissue sources. However, the short time window for derivation from donated fresh umbilical cord (UC) tissue fragments does not allow to consider biological features of the donor beyond serological safety testing. This limits the scope of MSC banking to rapid, prospective derivation of MSC, WJ lines without considering biological and genetic characteristics of the donor that may influence their suitability for clinical use (e.g. HLA type, inherited gene variants). In the present study, we describe a simple, efficient and reproducible approach for the cryopreservation of UC tissue fragments, compatible with established workflows in existing public frameworks for cord blood and tissue collection while guaranteeing pharmaceutical grade of starting materials for further processing under GMP standards. Herein we demonstrated the feasibility of time and cost-saving methods for cryopreservation of unprocessed UC tissue fragments directly at reception of the donated tissues using 10% Me2SO-based cryosolution and a commercial clinical-grade defined cryopreservation medium (Cryostor®), showing the preservation of all Critical Quality Attributes in terms of identity, potency and kinetic parameters. In summary, our study provides evidence that cryopreservation of large unprocessed UC tissue fragments (5-13.5 cm) supports subsequent progenitor cell isolation and derivation of MSC,WJ, preserving their viability, identity, proliferation rates and potency.

Tumor Suppressor Par-4 Regulates Complement Factor C3 and Obesity.

Prostate apoptosis response-4 (Par-4) is a tumor suppressor that induces apoptosis in cancer cells. However, the physiological function of Par-4 remains unknown. Here we show that conventional Par-4 knockout (Par-4-/-) mice and adipocyte-specific Par-4 knockout (AKO) mice, but not hepatocyte-specific Par-4 knockout mice, are obese with standard chow diet. Par-4-/- and AKO mice exhibit increased absorption and storage of fat in adipocytes. Mechanistically, Par-4 loss is associated with mdm2 downregulation and activation of p53. We identified complement factor c3 as a p53-regulated gene linked to fat storage in adipocytes. Par-4 re-expression in adipocytes or c3 deletion reversed the obese mouse phenotype. Moreover, obese human subjects showed lower expression of Par-4 relative to lean subjects, and in longitudinal studies, low baseline Par-4 levels denoted an increased risk of developing obesity later in life. These findings indicate that Par-4 suppresses p53 and its target c3 to regulate obesity.

Trophic niche of Australian cownose rays (Rhinoptera neglecta) and whitespotted eagle rays (Aetobatus ocellatus) along the east coast of Australia.

Australian cownose rays (Rhinoptera neglecta) and whitespotted eagle rays (Aetobatus ocellatus) are large myliobatiform rays that co-occur off temperate eastern Australia. Here, we performed stable-isotope analyses (δ13 C, δ15 N and δ34 S) on fin clips of both species to gain insights into their trophic interactions and isotopic niches, and assess the effect of preservation (ethanol-stored versus frozen) on isotopic values of fin-clip tissue of R. neglecta. Linear mixed models identified species as the main factor contributing to variation among δ15 N and δ34 S values, and disc width for δ13 C. Bayesian ecological niche modelling indicated a 57.4% to 74.5% overlap of trophic niches, with the niche of R. neglecta being smaller and more constrained. Because values of δ13 C were similar between species, variation in isotopic niches were due to differences in δ15 N and δ34 S values. Linear mixed models failed to detect differences in isotopic values of ethanol-stored and frozen fin tissue of R. neglecta. This study provides the first examination of the trophic ecology of R. neglecta and the comparison of isotopic niche with A. ocellatus, which will facilitate future research into the trophic interactions of these species and aid better resource management.

Bovine Satellite Cells Isolated after 2 and 5 Days of Tissue Storage Maintain the Proliferative and Myogenic Capacity Needed for Cultured Meat Production.

Cultured meat is an emerging alternative food technology which aims to deliver a more ethical, sustainable, and healthy muscle-tissue-derived food item compared to conventional meat. As start-up companies are rapidly forming and accelerating this technology, many aspects of this multi-faceted science have still not been investigated in academia. In this study, we investigated if bovine satellite cells with the ability to proliferate and undergo myogenic differentiation could be isolated after extended tissue storage, for the purpose of increasing the practicality for cultured meat production. Proliferation of bovine satellite cells isolated on the day of arrival or after 2 and 5 days of tissue storage were analyzed by metabolic and DNA-based assays, while their myogenic characteristics were investigated using RT-qPCR and immunofluorescence. Extended tissue storage up to 5 days did not negatively affect proliferation nor the ability to undergo fusion and create myosin heavy chain-positive myotubes. The expression patterns of myogenic and muscle-specific genes were also not affected after tissue storage. In fact, the data indicated a positive trend in terms of myogenic potential after tissue storage, although it was non-significant. These results suggest that the timeframe of which viable myogenic satellite cells can be isolated and used for cultured meat production can be greatly extended by proper tissue storage.

Lyophilization and homogenization of biological samples improves reproducibility and reduces standard deviation in molecular biology techniques.

Lyophilization is a cost-effective method for biological specimen preservation but detailed tissue-specific reference protocols are still lacking. Moreover, data are limited on the long-term stability of proteins and nucleic acids in lyophilized samples.Here, we offer lyophilization protocols for various rat and mouse tissues (kidney, heart, liver, lung, aorta, and skin) coupled with technical hints for optimal sample preparation. We demonstrate that lyophilized samples stored at 4 °C for 20 months can yield protein and RNA of similar quantity and quality to -80 °C storage, while phosphorylated proteins are preserved as well. Freeze-dried and subsequently pulverized samples can provide more consistent, more reliable data especially when investigating focal injuries, such as fibrosis. We developed a protocol for the concentration of biological solutions and achieved 20-times concentration in human peritoneal dialysis effluent solution which enables the previously unattainable detection of proteins in these samples. We established a method for water removal as well as accurate water content measurement of fecal samples, which can be valuable for gut metabolome analysis.Taken together, lyophilization is a valuable tool for the preservation of biological samples with many advantages. We aim to draw attention to the wide range of possibilities offered by freeze drying in pre-clinical or basic research.

Standardizing Proteomics Workflow for Liquid Chromatography-Mass Spectrometry: Technical and Statistical Considerations.

INTRODUCTION: The quantitative measurements based on liquid chromatography (LC) coupled with mass spectrometry (MS) often suffer from the problem of missing values and data heterogeneity from technical variability. We considered a proteomics data set generated from human kidney biopsy material to investigate the technical effects of sample preparation and the quantitative MS. METHODS: We studied the effect of tissue storage methods (TSMs) and tissue extraction methods (TEMs) on data analysis. There are two TSMs: frozen (FR) and FFPE (formalin-fixed paraffin embedded); and three TEMs: MAX, TX followed by MAX and SDS followed by MAX. We assessed the impact of different strategies to analyze the data while considering heterogeneity and MVs. We have used analysis of variance (ANOVA) model to study the effects due to various sources of variability. RESULTS AND CONCLUSION: We found that the FFPE TSM is better than the FR TSM. We also found that the one-step TEM (MAX) is better than those of two-steps TEMs. Furthermore, we found the imputation method is a better approach than excluding the proteins with MVs or using unbalanced design.

Back to Basics: Autologous Tissue: 1.5 www.aornjournal.org/content/cme.

Perioperative personnel manage autologous tissue when they care for patients undergoing procedures requiring the use of bone, soft tissue, or other autologous tissue to repair or replace defects. Use of autologous tissue can minimize the risk of rejection, disease transfer, and infection compared with the use of artificial materials. There are important steps to follow when handling autologous tissue to ensure it is safe for replantation and does not become contaminated. This Back to Basics article provides strategies for managing some types of autologous tissue, including bone flaps, parathyroid tissue, skin grafts, and veins. Tissue management strategies include creating strict documentation policies, standardizing processes and communication, and implementing routine audits to assess compliance.

A Team Approach to Improving Tissue Management.

Tissue implant management can be labor intensive because of multiple storage locations and cumbersome tracking systems. The purpose of this quality improvement (QI) project was to enhance patient safety and nursing satisfaction by upgrading our tissue-management facility and processes. We created a centralized storage room for tissue implants and staffed this room during all shifts. Tissue management was executed using tracking software and transportation devices that supported tissue receipt, storage, disposition, documentation, and reporting. Our project resulted in our full compliance with tissue implant requirements from the US Food and Drug Administration (FDA) and The Joint Commission. We also reduced our documentation error rate from 3% to less than 1%, and decreased the tissue-expiration rate by 1.1%. Tissues are now delivered to ORs, which allows RNs to focus on patient care rather than retrieval of implants. Monitoring of the tissue inventory has improved, resulting in the reduction of tissue wastage.

Refining the Diagnosis of Congenital Nephrotic Syndrome on Long-term Stored Tissue: c.1097G>A (p.(Arg366His)) WT1 Mutation Causing Denys Drash Syndrome.

Congenital nephrotic syndrome (CNS) caused by a mutation in the Wilms tumor 1 suppressor gene (WT1) is part of Denys Drash Syndrome or Frasier syndrome. In the framework of genetic counseling, the diagnosis of CNS can be refined with gene mutation studies on long-term stored formalin-fixed paraffin-embedded tissue from postmortem examination. We report a case of diffuse mesangial sclerosis with perinatal death caused by a de novo mutation in the WT1 gene in a girl with an XY-genotype. This is the first case of Denys Drash Syndrome with the uncommon missense c.1097G>A [p.(Arg366His)] mutation in the WT1 gene which has been diagnosed on long-term stored formalin-fixed paraffin-embedded tissue in 1993. This emphasizes the importance of retained and adequately stored tissue as a resource in the ongoing medical care and counseling.

Duration and method of tissue storage alters placental morphology - implications for clinical and research practice.

We assessed whether placental morphology is affected by placental storage before fixation. Fresh tissue from uncomplicated pregnancies (n = 10) was fixed immediately and further samples were stored dry, in PBS or culture medium for 24, 48 or 72 h at 4 °C. Placental morphology quantified using image analysis software found no difference in syncytial nuclear aggregates, cytokeratin 7, CD45 or Ki67 immunostaining irrespective of duration or mode of storage. The number of blood vessels per villus (CD31) was reduced in all conditions after 72 h (p < 0.05). Distal villous hypoplasia increased after 72 h (p < 0.05). Ideally, storage time should be minimised to ≤48 h prior to morphological or qualitative analysis.

Development of tissue bank.

The history of tissue banking is as old as the use of skin grafting for resurfacing of burn wounds. Beneficial effects of tissue grafts led to wide spread use of auto and allograft for management of varied clinical conditions like skin wounds, bone defects following trauma or tumor ablation. Availability of adequate amount of tissues at the time of requirement was the biggest challenge that forced clinicians to find out techniques to preserve the living tissue for prolonged period of time for later use and thus the foundation of tissue banking was started in early twentieth century. Harvesting, processing, storage and transportation of human tissues for clinical use is the major activity of tissue banks. Low temperature storage of processed tissue is the best preservation technique at present. Tissue banking organization is a very complex system and needs high technical expertise and skilled personnel for proper functioning in a dedicated facility. A small lapse/deviation from the established protocol leads to loss of precious tissues and or harm to recipients as well as the risk of transmission of deadly diseases and tumors. Strict tissue transplant acts and stringent regulations help to streamline the whole process of tissue banking safe for recipients and to community as whole.