Cryopreservation Methodologies Research Articles

Biopsy vitrification: New tool for endometrial tissue cryopreservation for research applications

Patient-derived endometrial biopsies serve as a crucial source for molecular studies, highlighting the necessity for tissue cryopreservation methods that preserve cell viability and tissue morphology with minimal to no impact. The passive slow freezing (PSF) protocol has demonstrated efficacy for cryopreserving endometrial biopsies, allowing for the subsequent isolation of viable epithelial and stromal cells. Vitrification (VT) enables the avoidance of ice crystal formation and could therefore potentially prevent mechanical injury to tissues. In this study, PSF and VT techniques were applied to endometrial biopsies, and the effects of cryopreservation on tissue samples were evaluated using traditional histology. In addition, transmission electron microscopy (TEM), gene expression profiling analyses, the viability of endometrial cells, and the ability to form epithelial organoids were compared between PSF and VT endometrial biopsies in a subset of samples. The histology and TEM studies demonstrated relatively mild cellular and sub-cellular damage in both cryopreservation protocols which did not affect tissue functionality and the formation of the organoids. Additionally, the cryopreservation methodology did not affect the gene expression profile of the 68 endometrial-receptivity associated genes studied. In conclusion, our findings indicate that although current cryopreservation methodologies need further improvements, they still allow us to achieve acceptable cell viability and functionality, showing promising potential for facilitating the utilization of cryopreserved endometrial tissue samples for research purposes.

Comparative transcriptome analysis reveals molecular damage associated with cryopreservation in Crassostrea angulata D-larvae rather than to cryoprotectant exposure

BackgroundThe Portuguese oyster Crassostrea angulata, a bivalve of significant economic and ecological importance, has faced a decline in both production and natural populations due to pathologies, climate change, and anthropogenic factors. To safeguard its genetic diversity and improve reproductive management, cryopreservation emerges as a valuable strategy. However, the cryopreservation methodologies lead to some damage in structures and functions of the cells and tissues that can affect post-thaw quality. Transcriptomics may help to understand the molecular consequences related to cryopreservation steps and therefore to identify different freezability biomarkers. This study investigates the molecular damage induced by cryopreservation in C. angulata D-larvae, focusing on two critical steps: exposure to cryoprotectant solution and the freezing/thawing process.ResultsExpression analysis revealed 3 differentially expressed genes between larvae exposed to cryoprotectant solution and fresh larvae and 611 differentially expressed genes in cryopreserved larvae against fresh larvae. The most significantly enriched gene ontology terms were “carbohydrate metabolic process”, “integral component of membrane” and “chitin binding” for biological processes, cellular components and molecular functions, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified the “neuroactive ligand receptor interaction”, “endocytosis” and “spliceosome” as the most enriched pathways. RNA sequencing results were validate by quantitative RT-PCR, once both techniques presented the same gene expression tendency and a group of 11 genes were considered important molecular biomarkers to be used in further studies for the evaluation of cryodamage.ConclusionsThe current work provided valuable insights into the molecular repercussions of cryopreservation on D-larvae of Crassostrea angulata, revealing that the freezing process had a more pronounced impact on larval quality compared to any potential cryoprotectant-induced toxicity. Additionally, was identify 11 genes serving as biomarkers of freezability for D-larvae quality assessment. This research contributes to the development of more effective cryopreservation protocols and detection methods for cryodamage in this species.

Opportunities and challenges related to sperm cryopreservation in Atlantic salmon gene banks

AbstractAtlantic salmon are facing population declines and loss of productivity within populations due to anthropogenic impact factors and reduced survival at sea. Biobanking is an increasingly used tool to conserve the genetic integrity and diversity of populations threatened by extirpation. The aim of the current article is to discuss the opportunities and challenges that increased use of cryopreservation brings to biobanking activities, using the Norwegian Gene Bank (NGB) for Atlantic salmon as a model system. The NGB was established in 1985 and involves a traditional living gene bank, as well as “frozen gene bank” where paternal germplasm is stored as cryopreserved sperm. Cryopreservation is a method where cells or tissues are frozen in liquid nitrogen to temperatures where all biological processes are paused, thus allowing the cells to remain viable after later warming/thawing to temperatures above 0°C. Cryopreservation is therefore used in long‐term preservation of genetic diversity and characteristics of wild populations. Until recently, implementation of large scale use of cryopreserved sperm in the live gene bank has been limited by a lack of protocols/capacity to preserve larger portions of sperms. More recent developments in cryopreservation methodologies, now enables preservation of samples sufficient for mass fertilization. Mass fertilization by cryopreserved sperm opens new opportunities to gene bank operations, including increased capacity to restore lost populations, mitigation of genetic changes in broodstock fish, as well as increased capacity at live gene bank facilities through the replacement of older males with frozen sperm. Knowledge demands regarding potential genetic damage to cryopreserved milt and potential epigenetic effects caused by the cryopreservation procedure should, however, be addressed.

Oocyte Cryopreservation at a Young Age Provides an Effective Strategy for Expanding Fertile Lifespan.

With an upward trend in delaying parenthood, women across the world face an increasing risk of age-related infertility and involuntary childlessness. Elective oocyte banking strategies offer women the possibility to protect part of their reproductive potential until personal finances, personal relationship, or career have stabilized. Timely collection and cryopreservation of oocytes when they are most competent and chromosomal abnormality rates have not yet escalated are crucial for achieving high live births through in vitro fertilization (IVF) treatment at a later stage. To promote reproductive autonomy, women shall be informed about the decrease in fertility rates that sharply intensifies from the age of 35 years and the strategies available to maintain their reproductive potential. Together with this information, women should also recognize the limitations of available strategies including expected live birth rates, costs of the procedures, and overall approach performance, which is mainly associated with age at cryopreservation, number of oocytes banked, and age at accessing the banked oocytes. Evidence-based statistics are not yet available due to the relatively short period in which oocyte cryopreservation has been offered for elective purposes and the scarce number of patients returning for accessing their oocytes. However, to evaluate the applicability of fertility cryopreservation on a large scale, several theoretical models have been proposed to assess the expected efficacy and overall cost-effectiveness of different oocyte banking strategies. In this study, we review current oocyte cryopreservation methodologies, their applications, and outcomes. Moreover, we summarize current evidence regarding known parameters affecting oocyte banking efficacy. Finally, we discuss key points that could play a role in improving access to the service and optimization of oocyte banking frameworks.

Fertility technologies and how to optimize laboratory performance to support the shortening of time to birth of a healthy singleton: a Delphi consensus.

To explore how the assisted reproductive technology (ART) laboratories can be optimized and standardized to enhance embryo culture and selection, to bridge the gap between standard practice and the new concept of shortening time to healthy singleton birth. A Delphi consensus was conducted (January to July 2018) to assess how the ART laboratory could be optimized, in conjunction with existing guidelines, to reduce the time to a healthy singleton birth. Eight experts plus the coordinator discussed and refined statements proposed by the coordinator. The statements were distributed via an online survey to 29 participants (including the eight experts from step 1), who voted on their agreement/disagreement with each statement. Consensus was reached if ≥ 66% of participants agreed/disagreed with a statement. If consensus was not achieved for any statement, that statement was revised and the process repeated until consensus was achieved. Details of statements achieving consensus were communicated to the participants. Consensus was achieved for all 13 statements, which underlined the need for professional guidelines and standardization of lab processes to increase laboratory competency and quality. The most important points identified were the improvement of embryo culture and embryo assessment to shorten time to live birth through the availability of more high-quality embryos, priority selection of the most viable embryos and improved cryosurvival. The efficiency of the ART laboratory can be improved through professional guidelines on standardized practices and optimized embryo culture environment, assessment, selection and cryopreservation methodologies, thereby reducing the time to a healthy singleton delivery.

Expansion of Human Dental Pulp Cells In Vitro Under Different Cryopreservation Conditions.

To optimize the expansion of human dental pulp cells in vitro by exploring several cryopreservation methodologies. The intra-dental pulp tissues from healthy subjects were extracted and divided into three separate tissue segments, which were randomly divided into the three following groups; the fresh group, the 5% DMSO group, and the 10% DMSO group. In the fresh group, dental pulp cells were directly cultivated as primary cultures, whereas in the DMSO groups, the dental pulp cells were cultivated from cryopreserved pulp tissue segments one month later. The cell yield and the time it took for the cells to grow out of the pulp tissue and attach to the culture plate varied among the three groups; the 5% DMSO group was inferior to the fresh group but superior to the 10% DMSO group (p<0.05). Moreover, no differences were found at the 1st passage amongst the three groups regarding the following aspects (p>0.05); colony formation rate and cell survival rate. Furthermore, no differences were noted at the 3rd passage regarding the following aspects (p>0.05); proliferation ability, cell growth curve and surface marker expression of dental pulp cells. Five percent DMSO may be the most optimal condition for tissue storage to preserve stem cells in situ.

In vitro conservation of Dendrobium germplasm.

Dendrobium is a large genus in the family Orchidaceae that exhibits vast diversity in floral characteristics, which is of considerable importance to orchid breeders, biotechnologists and collectors. Native species have high value as a result of their medicinal properties, while their hybrids are important as ornamental commodities, either as cut flowers or potted plants and are thus veritable industrial crops. Thus, preservation of Dendrobium germplasm is valuable for species conservation, breeding programs and the floriculture industry. Cryopreservation represents the only safe, efficient and cost-effective long-term storage option to facilitate the conservation of genetic resources of plant species. This review highlights 16 years of literature related to the preservation of Dendrobium germplasm and comprises the most comprehensive assessment of thorough studies performed to date, which shows reliable and reproducible results. Air-drying, encapsulation-dehydration, encapsulation-vitrification, vitrification and droplet-vitrification are the current cryopreservation methodologies that have been used to cryopreserve Dendrobium germplasm. Mature seeds, pollen, protoplasts, shoot primordia, protocorms and somatic embryos or protocorm-like bodies (PLBs) have been cryopreserved with different levels of success. Encapsulation-vitrification and encapsulation-dehydration are the most used protocol, while PLBs represent the main explant explored.

The use of a metal container for vitrification of mouse ovaries, as a clinical grade model for human ovarian tissue cryopreservation, after different times and temperatures of transport

Cryopreservation of ovarian tissue is paramount for fertility preservation, with important clinical applications, especially for women suffering from an oncological condition. Several cryopreservation methodologies have been tried in search of better outcomes, especially in terms of primor-dial and primary follicles integrity post-cryopreservation. Vitrification has successfully been applied to ovarian tissue using different carriers for tissue exposure to the liquid nitrogen (LN2). We developed an enclosed metal vessel, which has the advantage of a faster heat transfer, when in contact with LN2 avoiding at the same time, the direct contact with tissue. Additionally, we assessed the effect of different times and temperatures of transport between the collection of mouse ovaries and the beginning of cryopreservation, on follicular morphology after vitrification. Our results suggest that 37 °C and R.T. help to maintain normal primordial and primary follicle morphology for up to 4 hrs after collection and beginning of vitrification in a metal container. These data show that the metal container is an appropriate carrier for mouse ovary vitrification. The rate of morphologically normal primordial follicles up to 4 hrs.

Scalable culture and cryopreservation of human embryonic stem cells on microcarriers

As a result of their pluripotency and potential for unlimited self-renewal, human embryonic stem cells (hESCs) hold tremendous promise in regenerative medicine. An essential prerequisite for the widespread application of hESCs is the establishment of effective and efficient protocols for large-scale cell culture, storage, and distribution. At laboratory scales hESCs are cultured adherent to tissue culture plates; these culture techniques are labor-intensive and do not scale to high cell numbers. In an effort to facilitate larger scale hESC cultivation, we investigated the feasibility of culturing hESCs adherent to microcarriers. We modified the surface of Cytodex 3 microcarriers with either Matrigel or mouse embryonic fibroblasts (MEFs). hESC colonies were effectively expanded in a pluripotent, undifferentiated state on both Matrigel-coated microcarriers and microcarriers seeded with a MEF monolayer. While the hESC expansion rate on MEF-microcarriers was less than that on MEF-plates, the doubling time of hESCs on Matrigel-microcarriers was indistinguishable from that of hESCs expanded on Matrigel-coated tissue culture plates. Standard hESC cryopreservation methodologies are plagued by poor viability and high differentiation rates upon thawing. Here, we demonstrate that cryopreservation of hESCs adherent to microcarriers in cryovials provides a higher recovery of undifferentiated cells than cryopreservation of cells in suspension. Together, these results suggest that microcarrier-based stabilization and culture may facilitate hESC expansion and storage for research and therapeutic applications.

Influence of volume reduction and cryopreservation methodologies on quality of thawed umbilical cord blood units for transplantation

Introduction Although there is considerable variability in methodology among umbilical cord blood banks, their common goal is to achieve optimal product quality for transplantation. Cryopreservation is a critical issue for a long-term maintenance of cord blood viability and colony-forming capacities. Materials and methods We designed a prospective study to compare controlled (CRF) vs. non-controlled freezing (URF) of volume-reduced cord blood units. In addition, the influence of hydroxy ethyl starch (HES) on cryopreservation was also assayed. To assess the efficiency of protocols used, cell recoveries were measured and the presence of hematopoietic colony-forming units was quantified. Results In the study phase, we observed similar CB haematopoietc recoveries for CRF and URF strategies, except for TNC recovery that was better for HES volume reduced CB units in the URF group. When we analysed the data of routine processed CB units in samples from satellite cryovials, we found better BFU-E, CFU-GM, CFU-GEMM and CFU recoveries for those units processed with HES than without HES, in an URF manner. Conclusions URF of CB units is a cryopreservation procedure that allows similar hematopoietic progenitor recoveries than CRF with programmed devices. However, our study suggests that those banks that cryopreserve CB units in a URF manner should use HES for volume reduction. On the other hand, for CRF cryopreservation methodology volume reduction with and without HES are equally useful.

Cations in mammalian cells and chromosomes: Sample preparation protocols affect elemental abundances by SIMS

The focus of our current research aims at detailing and quantifying the presence of cations, primarily Ca and Mg, in mammalian cells and chromosomes throughout the different stages of the cell cycle, using our high resolution scanning ion microprobe, the UC-SIM. The 45 keV Ga + probe of this instrument, typically ∼40 nm in diameter, carries a current of 30–40 pA, appropriate for surface SIMS studies, but limited in sample erosion rate for dynamic SIMS mapping over cell-size areas, of order 100 μm × 100 μm. Practical and reliable use of this probe toward the above SIMS goals requires a careful matching of the latter factors with the physical and chemical consequences of sample preparation protocols. We examine here how the preferred sample cryo-preservation methodologies such as freeze-fracture and lyophilization affect high resolution SIMS analysis, and, from this standpoint, develop and evaluate the advantages and disadvantages of fast alternate approaches to drying frozen samples. The latter include the use of methanol, ethanol, and methanol/acetic acid fixative. Methanol-dried freeze-fractured samples preserve histological morphology and yield Ca and Mg distributions containing reliable differential dynamical information, when compared with those following lyophilization.

Ciba’s red pigment promotes photosynthesis

Orchids (Orchidaceae) are one of the most diverse plant groups on the planet with over 25,000 species. For over a century, scientists and horticulturalists have been fascinated by their complex floral morphology, pollinator specificity and multiple ethnobotanical uses, including as food, flavourings, medicines, ornaments, and perfumes. These important traits have stimulated world-wide collection of orchid species, often for the commercial production of hybrids and leading to frequent overexploitation. Increasing human activities and global environmental changes are also accelerating the threat of orchid extinction in their natural habitats. In order to improve gene conservation strategies for these unique species, innovative developments of cryopreservation methodologies are urgently needed based on an appreciation of low temperature (cryo) stress tolerance, the stimulation of recovery growth of plant tissues in vitro and on the ‘omics’ characterization of the targeted cell system (biotechnology). The successful development and application of such cryobiotechnology now extends to nearly 100 species and commercial hybrids of orchids, underpinning future breeding and species conservation programmes. In this contribution, we provide an overview of the progress in cryobanking of a range of orchid tissues, including seeds, pollen, protocorms, protocorm-like bodies, apices excised from in vitro plants, cell suspensions, rhizomes and orchid fungal symbionts. We also highlight future research needs.

A Molecular Basis of Cryopreservation Failure and its Modulation to Improve Cell Survival

The requirement for more effective cryopreservation (CP) methodologies in support of the emerging fields of cell bioprocessing and cell therapy is now critical. Current CP strategies appropriately focus on minimizing the damaging actions of physicochemical stressors and membrane disruption associated with extra- and intracellular ice formation that occurs during the freeze-thaw process. CP protocols derived from this conceptual paradigm, however, yield suboptimal survival rates. We now provide the first report on the identification of delayed-onset cell death following CP and the significance of modulating molecular biological aspects of the cellular responses (apoptosis) to low temperature as an essential component to improve postthaw outcome. In this study we quantitatively examined the molecular basis of cell death associated with CP failure in a canine renal cell model. In addition, we report on the significant improvement in CP outcome through the modulation of these molecular mechanisms by the utilization of an organ preservation solution. HypoThermosol. Further, the utilization of HypoThermosol as the preservation medium and the modulation of molecular-based cell death have led to a paradigm shift in biologic preservation methodologies. The recognition of molecular mechanisms associated with CP-induced cell death offers the promise of improved CP of more complex and/or fragile biological systems such as stem cells, engineered tissues, and human organs.

Validation of cryopreservation protocols for plant germplasm conservation: a pilot study using Ribes L.

Uniformly applicable techniques for germplasm preservation are important to the international genetic resources community and validation of techniques among working genebanks will enable the integration of new technologies into plant genetic resources programs. Apical meristems from micropropagated plants of Ribes nigrum L. cv. Ojebyn and R. aureum cv. Red Lake were used to test three cryopreservation protocols (controlled freezing, plant vitrification solution no. 2 (PVS2) vitrification and encapsulation–dehydration) at the USDA-ARS National Clonal Germplasm Repository (NCGR), Corvallis, OR, USA and the University of Abertay Dundee (UAD), Scotland. Similar results were obtained with PVS2 vitrification at both locations but meristem regrowth varied greatly for the other techniques. Variable results between the locations were noted for controlled freezing and were largely attributed to differences in ice crystal initiation by the controlled rate freezers. Low survival of `Red Lake' at UAD with all three techniques was attributed to poorly performing shoot cultures. Attention to protocol details is important for limiting variation between locations and step by step instructions for procedures and solution preparation aided protocol standardization. These studies suggest that source plant status, cryogenic facilities, and culture conditions may be the most likely causes of variation when validating cryopreservation methodologies in different locations. However, in-house optimization of standard procedures offers considerable potential in ensuring that cryopreservation methodologies can be transferred between international laboratories.

Cryopreservation of shoot-tips and meristems.

Shoot-tip meristem cryopreservation methodologies are reported for the complementary cryoprotective strategies of vitrification and equilibrium freezing using traditional controlled-rate freezing and chemical additive cryoprotection. Pregrowth, pretreatment, and cold acclimation approaches for the improvement of tolerance to liquid nitrogen are also presented. The chapter concludes by reporting an analytical protocol that profiles volatile hydrocarbon stress markers (for ethylene, hydroxyl radicals, and lipid peroxidation products) during cryopreservation. This method uses noninvasive headspace sampling and gas chromatography, and it is widely applicable across cryogenic systems.

Cryopreservation of Boar Semen

The present review summarizes information concerning the methods available to cryopreserve boar semen, covering the historical background, cryobiology and cryoprotecting considerations, technological developments and recent advances in cryopreservation methodologies. Successful methods for cryopreservation of boar semen have not been achieved despite numerous efforts world wide. Improvements in semen preservation technologies have been deterred by lack of in vitro methods that can accurately predict in vivo fertilizing capacity of frozen boar semen. The cell membrane is of crucial importance with regard to freeze-thaw survival of spermatozoa. It is important to optimize the survival of the plasma membrane as this is a non homogenous entity both in structure and function. The boar sperm membrane exhibits extreme sensitivity to freezing treatment. Freezing and thawing results in considerable changes in electrolyte dynamics and damages have mainly been associated with alterations in the head membranes especially at thawing. To date fruitless efforts have been carried out to find a cryoprotectant for the spermatozoa membranes and glycerol still continues to be used despite its harmful effects to the membranes.