High-throughput Cryopreservation Research Articles

Simulation analysis of high-throughput oyster cryopreservation at three scales of production.

Cryopreservation and germplasm repositories offer a variety of potential benefits to aquaculture industries. Despite this, no comprehensive repository systems exist for any prominent aquaculture species. A species that could greatly benefit from the use of cryopreserved sperm and repository storage is the eastern oyster, Crassostrea virginica. High-throughput cryopreservation protocols already exist for this species, and the easy transport of frozen sperm could facilitate selective breeding programs that address pressing challenges currently faced in the industry, such as mortality due to low-salinity conditions. This study addressed the gap between cryopreservation protocols and repository development in the oyster industry by creating simulation models to evaluate cryopreservation needs at three different scales of production. The effects of high-throughput device options and three key parameters (straws per oyster, batch size, and number of operators) on production capacity, time, and cost were evaluated. Recommendations for decisions concerning cryopreservation pathways and repository creation were given at each scale of production. Relative values of broodstock, juvenile oysters, and oyster sperm sold at hatcheries were also discussed. In general, repositories operating at higher production levels benefited from the economy of scale, could use automated high-throughput equipment options, and could hire more labor without drastically increasing production costs.

3D printed organisms for high-throughput cryopreservation

3D printed organisms for high-throughput cryopreservation

SIMULATION MODELING OF A HIGH-THROUGHPUT OYSTER CRYOPRESERVATION PATHWAY.

The genetic resources of oysters in Louisiana and the Gulf of Mexico are threatened due to high unexplained oyster mortality. Germplasm repositories are collections of cryopreserved genetic material stored alongside associated information that are used to protect genetics resources and facilitate breeding programs in agricultural industries. Therefore, there is great need for oyster repositories. Development of repositories has been slow despite research on high-throughput cryopreservation protocols because of logistical complexities. The goal of this study was to begin to address the gap between cryopreservation research and repository development in oyster aquaculture by modeling a cryopreservation protocol to understand and improve the process. The steps of a high-throughput cryopreservation protocol were defined and mapped in a process flow diagram. A simulation model was created using time study data, and key bottlenecks in the process were identified. Finally, model variations using alternate types of devices (tools or equipment) were created to address the identified bottlenecks. The model was found to accurately simulate the cryopreservation process. Parameters such as number of straws frozen per oyster, batch size, and number of operators significantly affected how the model performed and device choices produced substantial improvements. Simulation modeling has the potential to inform how cryopreservation pathways and repository systems in aquatic species should be structured and operated. There is ample opportunity for future work such as analyzing the impacts of production scale on cryopreservation processes.

High throughput cryopreservation of aquaculture seed

High throughput cryopreservation of aquaculture seed

Design and Cost Analysis of a Self-contained Mobile Laboratory for Commercial-scale Aquatic Species Cryopreservation.

Although aquatic species cryopreservation protocols have been studied around the world over the past 60 yr., germplasm repository development efforts and commercialization have begun only recently. The goal of this project was to develop a self-contained mobile laboratory for on-site high-throughput cryopreservation of aquatic species. The objectives of this study were to: (1) identify how a mobile laboratory would function in different operational scenarios, (2) customize an enclosed cargo trailer to function as a mobile laboratory, (3) evaluate the laboratory layout and ability of cryopreservation equipment to operate from generator power, and (4) document the investment costs for private and public groups to integrate a mobile laboratory into an existing cryopreservation facility at three levels of automation and estimate the total cost per trip based on hypothetical assumptions for two scenarios (aquaculture production and repository development). There were three operational designs identified for the mobile laboratory: (1) self-contained work inside the unit using generator power, (2) work inside the unit using external facility power, and (3) using the equipment inside of a host facility. The investment costs for a base-level mobile laboratory ranged between US$5670 and US$5787 for private groups and between US$5208 and US$5315 for public groups. With the addition of a range of automated processing equipment, total investment costs ranged from US$13,616 to US$103,529 for private groups and US$12,494 to US$94,891 for public groups. The total cost per trip to cryopreserve sperm of 59 blue catfish, Ictalurus furcatus, males to produce 6300 0.5-mL French straws was estimated to range from US$6089 to US$14,633 for private and between US$5703 and US$16,938 for public groups depending on the level of automation. Total cost per trip to cryopreserve sperm of 500 males of five different species in the genus Xiphophorus to produce 641 0.25-mL French straws was estimated to range from US$6653 to US$7640 for private and US$7582 to US$8088 for public groups depending on level of automation. Overall, a commercial-scale mobile laboratory was developed that can assist current germplasm activities and support future repository and industry development, and the layout information provided can help others to design and build comparable units.

A Strategy for Sperm Cryopreservation of Atlantic Salmon, Salmo salar, for Remote Commercial-scale High-throughput Processing.

Sperm cryopreservation is an essential tool for long-term storage of genetic resources for aquaculture fishes. The goal of this study was to develop an efficient and streamlined protocol for high-throughput processing for sperm cryopreservation in Atlantic salmon, Salmo salar. The objectives were to evaluate: 1) osmolality of blood serum for determining extender osmolality; 2) effects of extenders for fresh sperm dilution and refrigerated storage; 3) effects of methanol and dimethyl sulfoxide (DMSO) on fresh sperm motility, and 4) motility and fertility after thawing. In this study, sperm samples were collected at a hatchery site in Canada, and shipped to a freezing site located 2200 miles (3550 km) away in the United States. Evaluation of three extenders indicated that Mounib solution was suitable for diluting dry sperm for sample processing. Ten percent of methanol or DMSO was less toxic to sperm cells than was 15% within 30 min. Further testing with methanol at 5, 10, and 15%, and sperm solution:extender dilutions (v:v) of 1:1, 1:3, 1:19 (at concentrations of ~5×107; 3×108, and 1×109 cells/mL) indicated that methanol at 5% and 10% showed less toxicity to fresh sperm within 1 hr at sperm: extender dilutions of 1:1 and 1:3. Post-thaw motility of sperm cryopreserved with 10% methanol was significantly higher than that with 10% DMSO, and fertility reflected those results (0-1% in DMSO vs. 38-55% in methanol). Further evaluation of sperm cryopreservation with 10 and 15% methanol at sperm dilution ratios of 1:1, 1:3, 1:19 indicated post-thaw motility in 10% methanol was significantly higher than that in 15% methanol, and post-thaw fertility in 10% methanol at 1:1 and 1:3 dilution ratios had fertilization rates similar to that of fresh sperm controls. Sperm samples from 12 males cryopreserved with 10% methanol showed male-to-male variation in post-thaw motility (0-36%). Overall, a simplified standard protocol was established for cryopreservation of shipped sperm of Atlantic salmon using extender without egg yolk and yielded satisfactory post-thaw motility and fertilization rates. This procedure can be readily adopted by aquaculture facilities to take advantage of high-throughput cryopreservation capabilities at remote service centers. Most importantly, this approach lays the groundwork for an alternative commercial model for commercial-scale production, quality control and development of industrial standards. Control of male variability and sperm quality remain important considerations for future work.

3-D printing provides a novel approach for standardization and reproducibility of freezing devices

Cryopreservation has become an important and accepted tool for long-term germplasm conservation of animals and plants. To protect genetic resources, repositories have been developed with national and international cooperation. For a repository to be effective, the genetic material submitted must be of good quality and comparable to other submissions. However, due to a variety of reasons, including constraints in knowledge and available resources, cryopreservation methods for aquatic species vary widely across user groups which reduces reproducibility and weakens quality control. Herein we describe a standardizable freezing device produced using 3-dimensional (3-D) printing and introduce the concept of network sharing to achieve aggregate high-throughput cryopreservation for aquatic species. The objectives were to: 1) adapt widely available polystyrene foam products that would be inexpensive, portable, and provide adequate work space; 2) develop a design suitable for 3-D printing that could provide multiple configurations, be inexpensive, and easy to use, and 3) evaluate various configurations to attain freezing rates suitable for various common cryopreservation containers. Through this approach, identical components can be accessed globally, and we demonstrated that 3-D printers can be used to fabricate parts for standardizable freezing devices yielding relevant and reproducible cooling rates across users. With standardized devices for freezing, methods and samples can harmonize into an aggregated high-throughput pathway not currently available for aquatic species repository development.

A droplet-vitrification protocol enabled cryopreservation of doubled haploid explants of Malus x domestica Borkh. ‘Golden Delicious’

High throughput cryopreservation is a key step in the development of an apple t-DNA mutant library. The aim of this study was to develop an easy, efficient and rapid droplet vitrification protocol to cryopreserve doubled haploids of Malus x domestica Borkh. ‘Golden Delicious’. The doubled haploid genotype proved to be less tolerant to cryopreservation than ‘Golden Delicious’. Apices improved the quality of regeneration and the facility of dissection compared to meristems. About 50% of the explants treated with PVS2 for 40min survived after immersion in liquid nitrogen, and 10% showed sustainable development. Phytotoxicity was observed mostly after immersion in the vitrification solution. Hyperhydricity was regularly observed in these experiments and decreased final efficiency. The use of meta-topolin instead of 6-benzylaminopurine (BAP) reduced hyperhydricity by almost 50%. We demonstrated that doubled haploid apple apices can be cryopreserved, but further improvements are needed before this technique can be used at high throughput.

High throughput cryopreservation of cells by rapid freezing of sub-μl drops using inkjet printing--cryoprinting.

We have successfully used inkjet printing to cryopreserve 3T3 mouse fibroblast cells and human neuroprogenitor cells (NPCs) derived from human embryonic stem cells (hESCs). Sessile drops of volume 114 nl were formed by printing cell suspensions containing dimethyl sulphoxide (DMSO) as a cryoprotection agent (CPA) at rates in the region 100 Hz-20 kHz, from individual droplets of 380 pl. After printing and a freeze/thaw cycle (with a minimum 24 hours hold period at liquid N2 temperature), 3T3 cells showed an average viability of >90% with CPA concentration <0.8 M at all drop deposition rates. This is a significantly lower CPA concentration than normally used with conventional cryopreservation methods. Cell viability shows a small variation with the polymer substrates used, with the best results obtained using a polyimide substrate. The viability of 3T3 cells after 2 months storage at liquid nitrogen temperature was slightly reduced compared to the cells held for 24 hours but there was no significant further deterioration after 4 or 6 months storage. The viability of NPCs after an identical freeze/thaw cycle were only 55% but this is comparable with conventional cryopreservation methods that use much higher CPA concentrations. A parallel series of experiments printing cells onto substrates held at 195 K or directly into liquid N2 showed considerable variation in cell survival rate with drop deposition rate. Cell suspensions required higher levels of CPA than when printing followed by freezing. At low deposition rates a combination of DMSO and polyethylene glycol (PEG) was needed to allow cell viability after freezing. These results show that inkjet printing provides a practical high throughput method for the cryopreservation of cells with lower CPA concentrations than are required for current low volume cryopreservation methods.

On-site evaluation of commercial-scale hybrid catfish production using cryopreserved blue catfish sperm

Abstract Cryopreservation is an effective tool for conservation of genetic resources and is becoming increasingly used worldwide with aquatic species. Broadening the application of this technology to a commercial scale through high-throughput approaches has become essential for use with aquatic species. This study addressed high-throughput sperm cryopreservation of blue catfish at an industrial level. Our objectives were to: 1) optimize the sperm volume used for thawed sperm; 2) evaluate commercial application of high-throughput cryopreserved sperm with standard hatchery techniques, and 3) initiate evaluation of the fertility relationship between individuals and pooled samples. The results showed that a doubling of the previously established volume did not produce significant improvement in fertilization. The working volume of thawed sperm (2 ml at a concentration of 1 × 10 9 /ml for batches of 100–150 ml channel catfish eggs) was practical. There was no significant difference in fry production after artificial fertilization of 2 million eggs with cryopreserved or fresh sperm. Pooled sperm samples and the individual samples used to form the pools produced similar fertilization rates. Blue catfish sperm is valued as a genetic material for hybrid catfish production, and cryopreservation makes genetic material management possible. This study initiates industrialization of this technology for use with aquatic organisms, and because the technology can be generalized, expands the opportunities for application to other species. High-throughput cryopreservation of blue catfish sperm provides new capabilities and can maintain sperm quality sufficiently to support commercial hybrid production.

A quality assurance initiative for commercial-scale production in high-throughput cryopreservation of blue catfish sperm

Cryopreservation of fish sperm has been studied for decades at a laboratory (research) scale. However, high-throughput cryopreservation of fish sperm has recently been developed to enable industrial-scale production. This study treated blue catfish (Ictalurus furcatus) sperm high-throughput cryopreservation as a manufacturing production line and initiated quality assurance plan development. The main objectives were to identify: (1) the main production quality characteristics; (2) the process features for quality assurance; (3) the internal quality characteristics and their specification designs; (4) the quality control and process capability evaluation methods, and (5) the directions for further improvements and applications. The essential product quality characteristics were identified as fertility-related characteristics. Specification design which established the tolerance levels according to demand and process constraints was performed based on these quality characteristics. Meanwhile, to ensure integrity throughout the process, internal quality characteristics (characteristics at each quality control point within process) that could affect fertility-related quality characteristics were defined with specifications. Due to the process feature of 100% inspection (quality inspection of every fish), a specific calculation method, use of cumulative sum (CUSUM) control charts, was applied to monitor each quality characteristic. An index of overall process evaluation, process capacity, was analyzed based on in-control process and the designed specifications, which further integrates the quality assurance plan. With the established quality assurance plan, the process could operate stably and quality of products would be reliable.

High-Throughput Cryopreservation of In Vivo-Derived Swine Embryos

Cryopreservation of swine embryos is inefficient. Our goal was to develop a non-invasive method for “relatively” high-throughput cryopreservation of in vivo-produced swine embryos. Since removal of the lipid droplets within early swine embryos improves cryosurvival we wanted to apply a technique of high osmolality treatment followed by centrifugation that was first developed for in vitro-produced swine embryos to in vivo-produced swine embryos. The first aim was to determine how sensitive the in vivo-produced zygote and 2-cell stage embryo was to various high osmolality conditions for a short duration. Culture for 6, 12 or 18 min at 300, 400 or 500 milliosmoles (mOsm) had no detectable affect on the resulting blastocyst stage embryos (number of inner cell mass nuclei, trophectoderm nuclei, total number of nuclei, ratio of the trophectoderm to inner cell mass nuclei or percent blastocyst). However there was an effect of gilt on each of these parameters. For the second aim we focused on 300 mOsm for 6 min, 400 mOsm for 12 min, 500 mOsm for 12 min, and 500 mOsm for 18 min. The embryos were centrifuged for the duration of high osmolality treatment, then cultured to the blastocyst stage and vitrified. After vitrification and thawing the 500 mOsm for 18 min had the highest percent re-expansion with no difference in the total number of nuclei. While requiring a different base culture medium than in vitro-produced embryos, in vivo-derived embryos also survive cryopreservation without damage to their zona pellucida.

Simulation modeling of high-throughput cryopreservation of aquatic germplasm: a case study of blue catfish sperm processing.

Emerging commercial-level technology for aquatic sperm cryopreservation has not been modeled by computer simulation. Commercially available software (ARENA, Rockwell Automation, Inc. Milwaukee, WI) was applied to simulate high-throughput sperm cryopreservation of blue catfish (Ictalurus furcatus) based on existing processing capabilities. The goal was to develop a simulation model suitable for production planning and decision making. The objectives were to: 1) predict the maximum output for 8-hr workday; 2) analyze the bottlenecks within the process, and 3) estimate operational costs when run for daily maximum output. High-throughput cryopreservation was divided into six major steps modeled with time, resources and logic structures. The modeled production processed 18 fish and produced 1164 ± 33 (mean ± SD) 0.5-ml straws containing one billion cryopreserved sperm. Two such production lines could support all hybrid catfish production in the US and 15 such lines could support the entire channel catfish industry if it were to adopt artificial spawning techniques. Evaluations were made to improve efficiency, such as increasing scale, optimizing resources, and eliminating underutilized equipment. This model can serve as a template for other aquatic species and assist decision making in industrial application of aquatic germplasm in aquaculture, stock enhancement, conservation, and biomedical model fishes.

High-throughput cryopreservation of plant cell cultures for functional genomics.

Suspension-cultured cell lines from plant species are useful for genetic engineering. However, maintenance of these lines is laborious, involves routine subculturing and hampers wider use of transgenic lines, especially when many lines are required for a high-throughput functional genomics application. Cryopreservation of these lines may reduce the need for subculturing. Here, we established a simple protocol for cryopreservation of cell lines from five commonly used plant species, Arabidopsis thaliana, Daucus carota, Lotus japonicus, Nicotiana tabacum and Oryza sativa. The LSP solution (2 M glycerol, 0.4 M sucrose and 86.9 mM proline) protected cells from damage during freezing and was only mildly toxic to cells kept at room temperature for at least 2 h. More than 100 samples were processed for freezing simultaneously. Initially, we determined the conditions for cryopreservation using a programmable freezer; we then developed a modified simple protocol that did not require a programmable freezer. In the simple protocol, a thick expanded polystyrene (EPS) container containing the vials with the cell–LSP solution mixtures was kept at −30°C for 6 h to cool the cells slowly (pre-freezing); samples from the EPS containers were then plunged into liquid nitrogen before long-term storage. Transgenic Arabidopsis cells were subjected to cryopreservation, thawed and then re-grown in culture; transcriptome and metabolome analyses indicated that there was no significant difference in gene expression or metabolism between cryopreserved cells and control cells. The simplicity of the protocol will accelerate the pace of research in functional plant genomics.

Metaheuristic approaches to grouping problems in high-throughput cryopreservation operations for fish sperm

High-throughput cryopreservation operations of fish sperm is a technology being developed by researchers today. This paper first formulates a grouping problem in high-throughput cryopreservation operations of fish sperm and then develops a heuristic and four metaheuristic algorithms for its solution. The heuristic is modified from one originally proposed for the assembly line balancing problem. The four metaheuristic algorithms include simulated annealing (SA), tabu search (TS), ant colony optimization (ACO), and a hybrid differential evolution (hDE). For each metaheuristic algorithm, four different initialization methods were used. For both SA and TS, five different neighborhood solution generation methods were also studied. Real world data collected from a high-throughput cryopreservation operation was used to test the effectiveness of algorithms with different initialization and neighborhood solution generation methods. For comparison, a base line of grouping by processing order was also established. The results indicate that: (i) all algorithms performed better than the base line; (ii) using the result of the modified heuristic as the initial solution of metaheuristic algorithms lead to a better solution; the amount of improvement varied from algorithm to algorithm; (iii) among the five neighborhood solution generation operators, insertion operator was the best; (iv) among all algorithms tested, the hybrid differential evolution is the best, followed by tabu search in terms of average objective value.

Outlook for development of high-throughput cryopreservation for small-bodied biomedical model fishes

With the development of genomic research technologies, comparative genome studies among vertebrate species are becoming commonplace for human biomedical research. Fish offer unlimited versatility for biomedical research. Extensive studies are done using these fish models, yielding tens of thousands of specific strains and lines, and the number is increasing every day. Thus, high-throughput sperm cryopreservation is urgently needed to preserve these genetic resources. Although high-throughput processing has been widely applied for sperm cryopreservation in livestock for decades, application in biomedical model fishes is still in the concept-development stage because of the limited sample volumes and the biological characteristics of fish sperm. High-throughput processing in livestock was developed based on advances made in the laboratory and was scaled up for increased processing speed, capability for mass production, and uniformity and quality assurance. Cryopreserved germplasm combined with high-throughput processing constitutes an independent industry encompassing animal breeding, preservation of genetic diversity, and medical research. Currently, there is no specifically engineered system available for high-throughput of cryopreserved germplasm for aquatic species. This review is to discuss the concepts and needs for high-throughput technology for model fishes, propose approaches for technical development, and overview future directions of this approach.

Workshop and panel discussion: High-throughput cryopreservation of germplasm as an exchange currency for genetic resources

Workshop and panel discussion: High-throughput cryopreservation of germplasm as an exchange currency for genetic resources

Outlook for development of high-throughput cryopreservation for small-bodied biomedical model fishes

With the development of genomic research technologies, comparative genome studies among vertebrate species are becoming commonplace for human biomedical research. Fish offer unlimited versatility for biomedical research. Extensive studies are done using these fish models, yielding tens of thousands of specific strains and lines, and the number is increasing every day. Thus, high-throughput sperm cryopreservation is urgently needed to preserve these genetic resources. Although high-throughput processing has been widely applied for sperm cryopreservation in livestock for decades, application in biomedical model fishes is still in the concept-development stage because of the limited sample volumes and the biological characteristics of fish sperm. High-throughput processing in livestock was developed based on advances made in the laboratory and was scaled up for increased processing speed, capability for mass production, and uniformity and quality assurance. Cryopreserved germplasm combined with high-throughput processing constitutes an independent industry encompassing animal breeding, preservation of genetic diversity, and medical research. Currently, there is no specifically engineered system available for high-throughput of cryopreserved germplasm for aquatic species. This review is to discuss the concepts and needs for high-throughput technology for model fishes, propose approaches for technical development, and overview future directions of this approach.

High-throughput cryopreservation of spermatozoa of blue catfish ( Ictalurus furcatus): Establishment of an approach for commercial-scale processing

Hybrid catfish created by crossing of female channel catfish ( Ictalurus punctatus) and male blue catfish ( Ictalurus furcatus) are being used increasingly in foodfish aquaculture because of their fast growth and efficient food conversion. However, the availability of blue catfish males is limited, and their peak spawning is at a different time than that of the channel catfish. As such, cryopreservation of sperm of blue catfish could improve production of hybrid catfish, and has been studied in the laboratory and tested for feasibility in a commercial dairy bull cryopreservation facility. However, an approach for commercially relevant production of cryopreserved blue catfish sperm is still needed. The goal of this study was to develop practical approaches for commercial-scale sperm cryopreservation of blue catfish by use of an automated high-throughput system (MAPI, CryoBioSystem Co.). The objectives were to: (1) refine cooling rate and cryoprotectant concentration, and evaluate their interactions; (2) evaluate the effect of sperm concentration on cryopreservation; (3) refine cryoprotectant concentration based on the highest effective sperm concentration; (4) compare the effect of thawing samples at 20 or 40 °C; (5) evaluate the fertility of thawed sperm at a research scale by fertilizing with channel catfish eggs; (6) test the post-thaw motility and fertility of sperm from individual males in a commercial setting, and (7) test for correlation of cryopreservation results with biological indices used for male evaluation. The optimal cooling rate was 5 °C/min (Micro Digitcool, IMV) for high-throughput cryopreservation using CBS high-biosecurity 0.5-ml straws with 10% methanol, and a concentration of 1 × 10 9 sperm/ml. There was no difference in post-thaw motility when samples were thawed at 20 °C for 40 s or 40 °C for 20 s. After fertilization, the percentage of neurulation (Stage V embryos) was 80 ± 21%, and percentage of embryonic mobility (Stage VI embryo) was 51 ± 22%. There was a significant difference among the neurulation values produced by thawed blue catfish sperm, fresh blue catfish sperm ( P = 0.010) and channel catfish sperm ( P = 0.023), but not for Stage VI embryos ( P ⩾ 0.585). Cryopreserved sperm from ten males did not show significant variation in post-thaw motility or fertility at the neurulation stage. This study demonstrates that the protocol established for high-throughput cryopreservation of blue catfish sperm can provide commercially relevant quantities and quality of sperm with stable fertility for hybrid catfish production and provides a model for establishment of commercial-scale approaches for other aquatic species.

47. Towards a scalable, high-throughput cryopreservation strategy for human embryonic stem cells

47. Towards a scalable, high-throughput cryopreservation strategy for human embryonic stem cells