Addition Of Cryoprotectants Research Articles

Developing a simple and rapid method for cell-specific transcriptome analysis through laser microdissection: insights from citrus rind with broader implications

BackgroundWith the rapid development of single-cell sequencing technology, histological studies are no longer limited to conventional homogenized tissues. Laser microdissection enables the accurate isolation of specific tissues or cells, and when combined with next-generation sequencing, it can reveal important biological processes at the cellular level. However, traditional laser microdissection techniques have often been complicated and time-consuming, and the quality of the RNA extracted from the collected samples has been inconsistent, limiting follow-up studies. Therefore, an improved, simple, and efficient laser microdissection method is urgently needed.ResultsWe omitted the sample fixation and cryoprotectant addition steps. Instead, fresh samples were embedded in Optimal Cutting Temperature medium within 1.5 ml centrifuge tube caps, rapidly frozen with liquid nitrogen, and immediately subjected to cryosectioning. A series of section thicknesses of citrus rind were tested for RNA extraction, which showed that 18 μm thickness yielded the highest quality RNA. By shortening the dehydration time to one minute per ethanol gradient and omitting the tissue clearing step, the resulting efficient dehydration and preserved morphology ensured high-quality RNA extraction. We also propose a set of laser microdissection parameters by adjusting the laser power to optimal values, reducing the aperture size, and lowering the pulse frequency. Both the epidermal and subepidermal cells from the citrus rind were collected, and RNA extraction was completed within nine hours. Using this efficient method, the transcriptome sequencing of the isolated tissues generated high-quality data with average Q30 values and mapping rates exceeding 91%. Moreover, the transcriptome analysis revealed significant differences between the cell layers, further confirming the effectiveness of our isolation approach.ConclusionsWe developed a simple and rapid laser microdissection method and demonstrated its effectiveness through a study based on citrus rind, from which we generated high-quality transcriptomic data. This fast and efficient method of cell isolation, combined with transcriptome sequencing not only contributes to precise histological studies at the cellular level in citrus but also provides a promising approach for cell-specific transcriptome analysis in a broader range of other plant tissues.

Cryoprotective role of vacuum infused inulin on the quality of artichoke: Interactive effects of freezing, thawing and storage period

Freezing of artichoke is a promising alternative to storing it in brine and canning. The perishable vegetable was vacuum infused with inulin to improve freezing tolerance. Artichokes with and without inulin were frozen by static, air blast and individual quick freezing (IQF) methods and thawed by microwave, 25 °C and 4 °C temperature levels at each month of 6-months storage. Process conditions were evaluated by multivariate analysis of variance (MANOVA) and were found significant on the quality parameters. Inulin infusion better conserved the aw, color, texture, ascorbic acid and overall integrity of artichokes during frozen storage. Inulin incorporation and IQF showed mutual positive effect on drip loss. Polyphenol oxidase (PPO) activity values fitted to 2nd order kinetic and the highest residuals were determined in static freezing. PPO showed alleviating effect on total phenolic content. Vacuum impregnation caused a color difference prior to freezing, but was found effective for maintaining color during storage. As a result, the use of quick freezing techniques together with the addition of cryoprotectant was effective in the preservation of artichoke quality attributes during frozen storage.

Impact of various cryo-preservation steps on sperm rheotaxis and sperm kinematics in bull

Semen cryopreservation is an important tool that has massively contributed to the progression of animal reproduction, especially in cattle. Nonetheless, a large part of the sperm population suffers from cryostress and loses fertility during the process. Although bovine semen cryopreservation is more advanced than any other species, there are still some missing links in the technology knowledge. The aim of the current study was to detect the effect of cryopreservation steps on sperm rheotaxis. Semen samples were collected from sex bulls and analyzed inside a microfluidic platform with CASA after each step of cryopreservation, including control, dilution with yolk citrate, cryoprotectant addition, and cooling or freezing. The results showed that positive rheotaxis % (PR) was not affected during cryopreservation. On the contrary, the sperm kinematics of the positive rheotactic sperm undergo significant changes, as velocity parameters (VCL, VSL, and VAP) were lower in both the cryoprotectant adding and cooling/freezing steps than in the control and yolk citrate dilution steps, while progression parameters (LIN and BCF) were higher in the cryoprotectant and cooling/freezing steps than in the control and yolk citrate dilution steps. Beside these results, an interesting phenomenon of sperm backward positive rheotaxis has been observed. The results of backward sperm rheotaxis samples revealed a significant decrease in PR%, while all sperm kinematics except BCF were significantly higher than normal rheotaxis samples. Based on these results, we conclude that positive rheotactic sperm cells are the elite of the sperm population; however, they still get some sublethal cryodamage, as shown by alterations in sperm kinematics. We also suggest that the sperm-positive rheotaxis mechanism is a mixture of an active and passive process rather than a passive physical one.

Metabolites assay offers potential solution to improve the rooster semen cryopreservation

Semen cryopreservation represents a promising technology utilized for preserving high-quality chicken varieties in husbandry practices. However, the efficacy of this methodology is significantly impeded by the diminished quality of sperm. Metabolites, as the end products of metabolic reactions, serve as indicators of biological processes and offer insights into physiological conditions. In this study, we investigaged the sperm quality and alteration in metabolic profiles during the cryopreservation of Longyou Partridge Chicken semen. Following artificial semen collection, four groups of semen samples were established based on four points of the cryopreservation process (Ⅰ, fresh semen; Ⅱ, semen added extender and chilled at 4 °C for 30 min; Ⅲ, semen added cryoprotectants; Ⅳ, semen gradient freezed and stored in liquid nitrogen). Semen cryopreservation has a negative effect on the percentage of sperm in a straight-line trajectory (LIN), has no significant effect on total motile sperms (TM) or the proportion of sperm with typical morphology (NM). Metabolites were identified using LC-MS technique and analyses including Principal Component Analysis (PCA), Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA), Univariate statistical analysis, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were employed to identify metabolites. A total of 2471 metabolites had been identified, with the majority of the list being made up of amino acids and their metabolites as well as benzene and substituted derivatives. Group II exhibits 882 metabolites with significantly elevated abundance relative to Group I, alongside 37 metabolites displaying decreased abundance. In Group III, 836 metabolites demonstrate notably augmented abundance compared to Group II, while 87 metabolites exhibit reduced abundance. Furthermore, Group IV showcases 513 metabolites with markedly heightened abundance in comparison to Group III, and 396 metabolites with decreased abundance. Specific metabolites such as 5-Hydroxylysine, Phosphocholine, and alpha-d-glucose-6-phosphate exhibited a progressive decline during the cryopreservation process, correlating with either dilution and chilling, cryoprotectant addition, or freezing. In conclusion, our investigation systematically examined the changes of seminal metabolome and sperm quality throughout the cryopreservation process of rooster semen.

Farklı Şekillerde Hazırlanarak Değişik Sürelerde Depolanan Liyofilize Edilmiş ve Dondurulmuş Doğal Laktik Asit Bakteri Sıvılarının Laktik Asit Bakteri Sayılarının Belirlenmesi

In this study, it was aimed to determine the LAB numbers of fermented lactic acid bacteria (LAB) liquids prepared by adding different levels of sucrose and incubating for different periods of time by freezing in deep freezer and drying by lyophilization process and storing for one and three months. In the study, fermented LAB liquids prepared by adding different levels of sucrose (3%, 5% and 10%) and incubating for different periods (2, 5 and 10 days) were frozen with the addition of cryoprotectants (trisodiumsitrate (TRIS) and dimethylsulfoxide (DMSO)) and dried by lyophilization and stored for one and three months. While the highest LAB numbers in fermented LAB liquids frozen in deep freezer with TRIS and DMSO supplementation were obtained from 5.33x1010 cfu/ml group with 5% sucrose TRIS additive in 5 days incubation at the end of one month storage period, the highest LAB numbers were obtained from 8.33x109 cfu/ml group with 10% sucrose TRIS additive in 5 days incubation at the end of three months storage period (p

An effective strategy for improving the freeze-drying survival rate of Lactobacillus curvatus and its potential protective mechanism

Freeze-drying is a common method to prepare dried starter cultures with high stability and a long storage time. However, the bacterial cells suffer varying degrees of damage during the freeze-drying process. This study aimed to provide an effective strategy to improve the survival rate of Lactobacillus curvatus and to explore its potential protective mechanisms. The survival rates of bacterial cells in different cryoprotectants before and after freeze-drying were measured using the plate count method. The protective effects of composite cryoprotectants on bacterial cells were investigated from multiple perspectives, including key enzyme activities, the internal morphology of bacterial cells, and cell membrane integrity. Results showed that the freeze-drying survival rate of bacterial cells in the composite cryoprotectants consisting of trehalose, oleic acid and skimmed milk (85.38%) was significantly improved compared to that in PBS solution (2.04%). The addition of composite cryoprotectants could reduce damage to the cell wall and cell membrane, maintain the normal morphology of bacterial cells and cell membrane integrity, and improve key enzyme activities (lactate dehydrogenase, pyruvate kinase and ATPases) during the freeze-drying process. The viability of freeze-dried cultures was also monitored at different temperatures during storage, and the cell survival of freeze-dried cultures at −20 °C was the highest, reaching 61.13%. The findings obtained in this work offer a new method to improve the viability of Lactobacillus curvatus after freeze-drying, and may promote the wide use of freeze-dried cultures in the food fermentation industry.

Влияние криопротекторов на изменение состава и свойств рыбного фарша при его холодильной обработке

Modern technologies of cryopreservation of biological raw materials provide for the use of cryoprotectors that act as regulators of the ice crystallization process and affect denaturation changes in the protein structures of bio-logical objects. For fish raw materials, the change of these two technological factors ensures the reversibility of the freezing process, i.e. the quality of the product after processing, as well as production losses during its implementation. Substances belonging to various classes of chemical compounds are used as cryoprotectors, but cryoprotectors traditionally used in cryopreservation - alcohols, oxides (DMSO) and carbohydrates – cannot be widely used in food production due to their toxicity and sweet taste. Therefore, the search for effective cryoprotectors intended for refrigeration technologies of food is an important task of scientific and industrial orientation. The aim of the work was to experimentally prove the feasibility of using food additives - cryoconcentrates of seafood – as cryoprotectors in the refrigeration technology of fish products. When realizing the purpose of the work, the tasks of studying changes in the composition and properties of minced fish during its refrigeration processing and assessing the quality and storage capacity of newly developed molded products with cryoprotectors were solved. Chilled pollock, which has a shelf life of no more than two days, was used as the main raw material for the production of minced fish and products based on it. A mixture of dry seafood concentrates “Mineralocorrecting” and dry concentrate of Pacific herring milk were used as natural cryoprotective substances. Sorbitol, table salt, pectin, glycerin were also used in the compositions of cryoprotective additives. It was found that the introduction of cryoprotective compositions into minced fish reduces the loss of tissue juice of minced fish by various technological operations of refrigeration processing by more than 2 times. Freezing and cold storage leads to a weakening of the structural and functional properties of minced fish. More pronounced negative changes in the initial properties of minced fish are noted in the control sample; in the minced meat containing the composite cryoprotectors developed by us, they manifest themselves to a much lesser extent. According to the sensory evaluation of minced fish samples, the quality during refrigeration is somewhat reduced to a greater extent in the control sample, to a lesser extent in samples containing cryoprotectors. It should be noted the high quality and storage capacity of new fish molded products with cryoprotective additives for 6 months.

Оптимизация состава криопротектора для сохранения репродуктивных клеток африканского сома

The article presents a study on the selection of the optimal composition of the protective medium for cryopres-ervation of the reproductive cells of male African catfish. Glycerol, dimethyl sulfoxide (DMSO) at concentrations of 3, 5 and 10% were studied as the main cryoprotective additives. The influence of these compounds on the quality indicators of catfish sperm (the percentage and time of sperm movement after activation), the level of sperm lipid peroxidation, and the activity of the antioxidant enzyme catalase before and after the cryopreservation process was assessed. A decrease in fish breeding indicators of the quality of African catfish sperm (the proportion of living cells, motility time) was established when DMSO was added to the basic cryomedium. The greatest decrease in the percentage of sperm motility before freezing is observed when DMSO is added at a concentration of 10%. A dose-dependent effect of a decrease in sperm activity after freezing was noted with an increase in the addition of DMSO to the cryogenic medium from 3 to 10%. It has been shown that 3% glycerol in a cryogenic medium does not contribute to the preservation of cells during freezing, cryodamage caused by the formation of ice crystals leads to a significant decrease in the motility of catfish sperm. The best indicators of sperm quality were obtained when using glycerol, the most effective concentration is 5% (40% of mobile reproductive cells within 53 s). The addition of 5% glycerol to the cryomedium contributes to the greatest decrease in the level of peroxidation of sperm lipids and an increase in catalase activity, which is fully consistent with the results on sperm motility. The calculated correlation coefficients confirm the dependence of the reproductive qualities of gametes on the antioxidant status of cells. The results indicate the importance of using antioxidants as cryoprotective additives to improve the quality of African catfish sperm breeding and indicate the need for more research to evaluate the effect of adding new potentially effective and safe antioxidants as cryoprotective additives.

Isochoric Supercooling Organ Preservation System.

This technical paper introduces a novel organ preservation system based on isochoric (constant volume) supercooling. The system is designed to enhance the stability of the metastable supercooling state, offering potential long-term preservation of large biological organs at subfreezing temperatures without the need for cryoprotectant additives. Detailed technical designs and usage protocols are provided for researchers interested in exploring this field. The paper also presents a control system based on the thermodynamics of isochoric freezing, utilizing pressure monitoring for process control. Sham experiments were performed using whole pig liver sourced from a local food supplier to evaluate the system's ability to sustain supercooling without ice nucleation for extended periods. The results demonstrated sustained supercooling without ice nucleation in pig liver tissue for 24 and 48 h. These findings suggest the potential of this technology for large-volume, cryoprotectant-free organ preservation with real-time control over the preservation process. The simplicity of the isochoric supercooling device and the design details provided in the paper are expected to serve as encouragement for other researchers in the field to pursue further research on isochoric supercooling. However, final evidence that these preserved organs can be successfully transplanted is still lacking.

An improvement to capture and visualize the protein conformational ensembles in double electron-electron resonance measurements.

Over the past decades, pulsed ESR double electron-electron resonance (DEER) has emerged as an effective biophysical tool for revealing protein conformational ensembles. With a collection of numerous interspin distances by DEER, they can be utilized to reveal the dynamic structural ensembles as opposed to static structures from other techniques. In a continuing effort to improve the technique, we present a combined approach of experimental and simulation advances that allows one to capture and visualize protein conformational ensembles in the absence of cryoprotectant additives. First, we present a rapid freezing method using a flexible fused silica microcapillary, enabling DEER measurement without the use of cryoprotectants. Second, we present a Monte Carlo based algorithm that allows one to efficiently utilize the sparse DEER data for deriving ensemble structures of a target protein. The Bid protein, a highly glycerol-sensitive protein, is used as a model. We show that our rapid-freezing DEER method can capture the Bid ensembles in the absence of glycerol, which was not possible with the conventional DEER setup. With the newly developed algorithm, we can efficiently visualize the Bid conformational ensembles, saving a lot for performing advanced structural modeling. Our combined method advances DEER for capturing and visualizing protein conformational ensembles.

Development of cryopreservation media for the slow-freezing of cultured primordial germ cells in chicken.

Conservation of chicken germplasm is crucial in supporting commercial breeds for sustainable egg and meat production and preserving the genetic diversity of indigenous breeds for future breeding. Cryopreservation of chicken fertilized eggs or embryos is not feasible, owing to the large yolk-laden structure of the eggs. Primordial germ cells (PGCs), the embryonic precursors of gametes, are the best candidates for the cryobanking of chicken germplasm. Effective cryobanking of chicken PGCs requires an optimal cryopreservation protocol. Cryomedia containing dimethyl sulfoxide (DMSO) or DMSO combined with serum have been widely used for the cryopreservation of chicken PGCs. However, as cryoprotectants are yet to be optimized for chicken PGCs, the efficacy of cryomedia can be further improved. Here, we investigated the cryoprotective effects of propylene glycol (PG), an alternative to DMSO, on chicken PGCs. We found that the addition of non-permeable cryoprotectants, such as trehalose or chicken serum, to DMSO or PG cryomedia improved the recovery and survival rates of post-thawed PGCs. We further investigated the cryoprotective effects of trehalose and chicken serum and found that these additives have different cryoprotective actions. Based on these findings, we designed two different cryomedia: DTS, including 5% DMSO, 0.3 M trehalose, and 1% chicken serum, and PTS, including 7.5% PG, 0.1 M trehalose, and 5% chicken serum. Among the different PGC lines and freshly isolated PGCs, the cryomedia showed similar post-thaw recovery rates. Following transplantation, post-thawed male PGCs can colonize gonads and differentiate into functional sperm. We successfully revived the offspring of Kurokashiwa, a rare chicken breed in Japan, with cryopreserved PGCs. In conclusion, we developed two different cryomedia that achieved > 50% recovery of viable PGCs after thawing while maintaining germline competency.

Comparison of Commercial Poultry Semen Extenders Modified for Cryopreservation Procedure in the Genetic Resource Program of Czech Golden Spotted Hen.

Simple SummaryThe Czech Golden Spotted Hen is the only original Czech hen breed included in the National Genetic Reserve Program. The objective of this program is to preserve the genetic resources of native breeds in the Czech Republic through reproductive biotechnologies, such as artificial insemination or the cryopreservation of sperm. In this study, three commercial liquid storage extenders supplemented with a 9% N-methylacetamide cryoprotectant were investigated. Using a defined methodology, modified Poultry media® and Raptac® extenders were identified as promising tools for the cryopreservation procedure. Thus, an important strategy for the National Genetic Reserve Program of the Czech Golden Spotted Hen was suggested.Spermatozoa cryoconservation represents an important strategy for partial in vitro or rescue programs designed for threatened livestock populations. The procedure for the semen cryopreservation of the Czech Golden Spotted Hen was proposed due to the lower fertilization rate of poultry semen compared to mammalian species. The aim of this study was to compare commercial extenders designed for liquid storage preservation with the use of a predefined cryoprotectant, and, thus, to propose an important tool for the procedure of the semen cryopreservation of the Czech Golden Spotted Hen. Ejaculates were sampled from four roosters during five semen collection days. The samples were frozen in Poultry media®, Raptac® and NeXcell® extenders supplemented with a 9% N-methylacetamide (NMA) cryoprotectant. Sperm parameters of the total motility (MOT; %), plasma membrane and acrosome intactness (PAI; %), plasma membrane damage (%), acrosome damage (%) and cells with plasma membrane and acrosome damage (%) were assessed using a mobile mCASA analyzer and flow cytometer after the cryopreservation of the insemination doses (IDs). For Poultry media® (PAI = 51.11%; MOT = 23.58%) and Raptac® (PAI = 52.04%; MOT = 23.13%) extenders with the addition of an NMA cryoprotectant, the comparable results were detected after thawing. For NexCell® media, the results were poor (PAI = 7.07%; MOT = 3.83%). Our results indicated two extenders suitable for the cryopreservation procedure, with the applied modification.

Low-temperature vacuum permeation of sodium tripolyphosphate and trehalose suppresses the denaturation of myofibrillar proteins in peeled shrimp (Litopenaeus vannamei) during frozen storage.

Phosphates and trehalose are widely accepted additives in animal muscle products. In this study, the effects of pre-soaking with sodium tripolyphosphate (STPP) and trehalose under vacuum permeation (VP) conditions on the physicochemical properties of shrimp muscle were evaluated over 120 d of frozen storage. The results indicate the STPP/trehalose-VP treatments significantly reduced the thawing loss and prevented changes in the texture, myofibrillar protein (MP) content, and Ca2+-ATPase activity of shrimp muscle during frozen storage compared with results of control and individual STPP or trehalose soaking treatments. The histological structure analysis revealed the permeated STPP/trehalose distinctly inhibited the dissociation of muscle fibers and reduced physical damage to connective tissues during storage. Furthermore, analysis of the thermal properties indicated STPP/trehalose treatment increased the values of shrimp muscle tissues, likely by restricting the mobility of water molecules in muscle tissues and embedding proteins in the glassy matrix. Thus, the physical destruction caused by ice crystal growth was greatly reduced, due to the absence of water molecules around muscle proteins during frozen storage. Accordingly, the combined STPP/trehalose-VP treatment significantly enhanced the stability of frozen shrimp, and the results support the application of traditional cryoprotective additives. The treated shrimp can be stored at comparatively higher temperatures with limited physicochemical reactions during frozen storage.

The Effect of Cryopreservation on the Survival of Nocardia farcinica and Yersinia pestis vaccine strains.

Pathogenic microorganisms are valuable biological resources, closely related to biosecurity, human health, environmental protection, and renewable energy. It is very important to properly preserve the microbial resources by methods to maintain the purity, viability, and integrity, and to avoid prolonged degradation. The present work aims to explore the cryopreservation technology of Nocardia farcinica (Gram-positive bacteria) and Yersinia pestis vaccine strains (Gram-negative bacteria). The effects of cryoprotectants (CPAs), freezing temperature, and freeze-thaw cycles on the two bacteria in the cryopreservation process were studied. The results showed that the addition of CPAs (glycerol, propylene glycol, sucrose, glucose, l-carnitine, l-proline, and skim milk) significantly enhanced the survival rates of the N. farcinica and Y. pestis vaccine strains. However, high concentrations of CPAs can produce biochemical toxicity in the two pathogens. The utilization of composite CPAs not only reduced the toxicity but also improved the survival rates of samples during cryopreservation. The optimal composite CPA for N. farcinica is 0.292 M sucrose, 0.62 M l-carnitine, and 2.82 M glycerol. The optimal composite CPA for Y. pestis is 0.62 M l-carnitine, 8.46 M glycerin, and 0.292 M sucrose. The results showed that the quality of the strains stored at -80°C and -196°C was better. For the case of freeze-thaw cycles, the two pathogens have different degrees of reduction, and the survival rate of Y. pestis decreased more than that of N. farcinica. The uniform distribution of bacteria in CPAs can form uniform nucleation sites in the solution system, which is beneficial to the cryopreservation of strains, as can be seen from the experimental results from a differential scanning calorimeter. This study may provide a reference for better preservation of precious natural biological resources of pathogenic microorganisms.

Probiotics viability in frozen food products

Improving gastrointestinal flora with probiotics is a promising method of treating and preventing many diseases. However, in food industry, introduction of probiotics is limited by the shelf life of products, as well as by the reduction of bacterial viability during storage. Thus, introduction of probiotics into foods to be frozen is a promising method to extend the shelf life of probiotic products. However, the storage of living cells in frozen products involves their damage during freezing and subsequent storage at temperatures below 0 °C. Therefore, some technological solutions are required to protect probiotics during storage at sub-zero. Today, encapsulation, addition of cryoprotectants and prebiotics, as well as adaptation of microorganisms are widely used techniques of protecting probiotics. The combination of various protection methods is a promising way to improve the viability of probiotics during storage. Studies aimed at investigating various combinations of hurdle methods and their effect on bacterial survival are of great interest.This review compiles the most common methods of preserving probiotics in frozen foods, describing their effects, variety of approaches, and effectiveness. The main mechanisms of bacterial cell damage by freezing are described. Further trends in probiotic protection in frozen foods are also discussed.

Temperature-pressure correlations of cryoprotective additives for the design of constant volume cryopreservation protocols

In the recent years, the use of constant volume (isochoric) cryopreservation, in medicine and biotechnology has captured more attention from the research community and now there is an increasing interest in the use of this new technology. It has been established that the thermodynamics of isochoric freezing is different from that of isobaric (constant pressure) freezing. This study provides researchers in the field experimental results for various compositions of cryoprotectants commonly used in isobaric cryopreservation, in terms of temperature-pressure-molar concentration correlation. It also reveals experimental isochoric thermodynamic data for the following cryoprotectants, commonly used in isobaric cryopreservation: dimethyl sulfoxide, trehalose, ethylene glycol and diethylene glycol. Currently, the data on the pressure-temperature correlation in an isochoric system of cryoprotectants used in isobaric cryopreservation is not available. Our new experimental results indicate that the studied concentrations for each of the CPAs, lower and expands the range of temperatures in which cryopreservation by isochoric freezing can be safely practiced. We consider that these experiments will aid researchers developing new isochoric cryopreservation protocols.

Automated closed volume reduction process for apheresis stem cell grafts: From development to clinical implementation.

Collection of HPC by apheresis (HPC-A) can sometimes result in higher collection volumes, increasing the dimethyl sulfoxide (DMSO) volume infused into patients and the space requirements in liquid nitrogen freezers. Volume reduction prior to the addition of cryoprotectant is an efficient means to reduce the DMSO load infused into patients and to optimize freezer storage space. To implement a closed semi-automated volume reduction process, a method was developed to produce leukocyte-rich mock apheresis products using buffy coats derived from whole blood collections. The mock HPC products were then used to measure the efficiency and reliability of the semi-automated process over a range of volumes and cell concentrations. The resulting data was used to support the implementation of the process with concurrent monitoring. A closed, semi-automated volume reduction process resulted in recoveries of over 93% and 91% of white blood cells and CD34+ cells with no significant loss of product viability or potency. Mean doses of CD34+ and CFU infused per kilogram recipient body weight were 4.0 ± 1.1 × 106 /kg and 4.2 ± 1.7 × 105 /kg, resulting in no delays in median time to neutrophil and platelet engraftment, significant increase in adverse reaction or nonconformances. The effectiveness outcomes of the first Canadian experience in the implementation of a closed semi-automated volume reduction system in the processing of HPC-A products for autologous transplant have met the predetermined acceptance criteria, supporting its use in a stem cell manufacturing laboratory compliant with good manufacturing practice regulations.

P-242 Oocyte morphology before vitrification is associated with the post-warming survival rate

Abstract Study question Although oocyte vitrification is efficient for fertility preservation, the information about damage post-warming is limited. Could the oocyte morphology before vitrification predict the survival rate? Summary answer Precisely measuring the oolemma/whole oocyte surface area ratio may predict the post-warming survival rate before performing oocyte vitrification. What is known already Recent studies suggest that oocyte vitrification has higher post-warming survival and pregnancy rates. However, the oocyte post-warming survival rate depends on the oocyte quality but not maternal age. Furthermore, it has been well received that the cell surface/volume ratio is related to the efficiency of cryoprotective additives permeability. Moreover, the oocyte shows a large perivitelline space (PVS) that is negatively related to clinical outcomes. Nevertheless, no studies precisely defined large PVS by measuring the oolemma/whole oocyte surface area ratio. Therefore, we assessed the correlation between the oolemma/whole oocyte surface area ratio and the post-warming survival rate. Study design, size, duration A retrospective study enrolled 43 IVF cycles undergoing oocyte thawing from 2018 to 2020 at Linkou Chang Gung Memorial Hospital. Participants/materials, setting, methods A total of 161 oocytes were warmed. We excluded the oocyte were shipped to our center, MI stages, and without electric medical images. We retrospectively collected all the electric medical images of warmed oocytes that were measured the surface area of oolemma and whole oocyte using ImageJ. Linear regression and Receiver operating characteristic (ROC) curve analysis were performed for further analysis. Main results and the role of chance Our finding showed that the maternal age (38.1 ± 5.3) at oocyte vitrification was not correlated to survival rate (P = 0.26) and the oolemma/whole oocyte surface area ratio (P = 0.59). However, the ROC curve revealed that the oolemma/whole oocyte surface area ratio could predict the survival rate post-warm (AUC: 0.67, 95%CI: 0.56-0.78). Limitations, reasons for caution Small sample size limited the power of this study, and the higher advanced maternal age group involved in this study may not represent the younger group’s outcomes. Therefore, future studies need to enlarge the sample size to evaluate the prediction efficiency of the oolemma/whole oocyte surface area ratio. Wider implications of the findings Our study demonstrates the possible factor related to oocyte cryo-damage. The lower oolemma/whole oocyte surface area ratio represents the oocyte having a larger PVS that has a higher chance of damage after thawing, which shows that by precisely qualified the size of PVS could evaluate the oocyte quality before vitrification. Trial registration number Not applicable

Recovery of spindle morphology and mitochondrial function through extended culture after vitrification-warming of bovine oocytes

The present study aimed to determine the effects of vitrification on the meiotic spindle and mitochondrial function of bovine oocytes submitted to different times of post-warming culture. Partially denuded cumulus-oocyte complexes were vitrified at different maturation times (18-, 20-, and 24-h) using a two-step cryoprotectant addition protocol and submitted to 6-, 4-, or 0-h of post-warming extended culture in maturation medium. Microtubule configuration and chromosomal arrangement were analyzed after 0- and 6-h of extended culture, whereas mitochondrial membrane potential and ATP content were measured at 0-, 4-, and 6-h of post-warming recovery. Results of meiotic spindle integrity revealed that vitrified-warmed oocytes that underwent 6-h of culture had similar incidence of normal microtubule configuration and chromosomal arrangement as compared to fresh oocytes, but higher than oocytes in the vitrification control group (no culture). Mitochondrial membrane potential was not different in all the vitrification groups, but the oocytes that were cultured for 4-h after warming had similar levels compared to fresh oocytes. ATP concentration in all vitrification groups was lower than the control group. However, oocytes cultured for 6-h had the lowest rate of ATP depleted oocytes among the vitrification groups. The results of this study indicate that extended culture after warming promotes the recovery of the meiotic spindle and, to some extent, mitochondrial function of vitrified-warmed metaphase II bovine oocytes.

Efficacy of the incorporation between self-encapsulation and cryoprotectants on improving the freeze-dried survival of probiotic bacteria.

This study aimed to improve the viability of probiotic bacteria during freeze-drying by the combination of self-encapsulation and cryoprotectants. Lactiplantibacillus plantarum VAL6 and Lactobacillus acidophilus VAR1 were exposed to environmental stresses including temperature, pH and increased CO2 concentration before performing freeze-drying with the addition of cryoprotectants. The results proved that tested stresses can stimulate the bacteria to synthesize more extracellular polymeric substances to form self-encapsulation that increases their freeze-dried viability. In combination with cryoprotectants to form double-layered microencapsulation, L. plantarum VAL6 stressed at pH 3.5 in combination with whey protein isolate could achieve the highest Improving Cell Viability of 4361-fold, while L. acidophilus VAR1 stressed at 25o C in combination with alginate gave a maximum Improving Cell Viability of 73.33-fold. The combination of self-encapsulation and cryoprotectants significantly improves the freeze-dried viability of probiotics. This is the first report that uses environmental stress to stimulate extracellular polymeric substance synthesis for self-encapsulation formation combined with the addition of cryoprotectants to enhance the freeze-dried survival of probiotics. This could be a novel approach in improving the viability of probiotic strains for various applications.