A Modular Open-Technology Device to Measure and Adjust Concentration of Sperm Samples for Cryopreservation

Repositories for aquatic germplasm can safeguard the genetic diversity of species of interest to aquaculture, research, and conservation. The development of such repositories is impeded by a lack of standardization both within laboratories and across the research community. Protocols for cryopreservation are often developed ad hoc and without close attention to variables, such as sperm concentration, that strongly affect the success and consistency of cryopreservation. The wide dissemination and use of specialized tools and devices can improve processing reliability, provide data logging, produce custom hardware to address unique problems, and save costs, time, and labor. The goal of the present work was to develop a low-cost and open-technology approach to standardize the concentration of sperm samples prior to cryopreservation. The specific objectives were to: 1) fabricate and test a peristaltic pump and optical evaluation module (POEM); 2) fabricate and test a prototype of the modular, open-technology concentration measurement and adjustment system (CMAS), which incorporated the POEM; 3) identify opportunities to extend the CMAS to microliter volumes through low-cost resin 3-D printing, and 4) identify strategies from this work that can be applied to future open fabrication efforts. The POEM and CMAS were prototyped and tested with biological samples. A relationship between optical signal and cell concentration of channel catfish (Ictalurus punctatus) sperm samples was established by linear regression. In a blind trial, cell concentrations were estimated with the POEM and correlated closely to their known concentrations (linear regression R2 = 0.9945) in a range of 1 × 108 to 4 × 109 cells/mL. The CMAS was able to estimate and adjust the concentration of a sample of the marine microalgae Tetraselmis chuii as a preparatory step for cryopreservation. To explore the possible use of the CMAS with microliter sample volumes in future work, evaluation of low-cost resin 3-D printing showed that this technology can approach conventional microfabrication techniques in feature quality and resolution. The development of the CMAS as open technology can provide opportunities for community-level standardization in cryopreservation of aquatic germplasm, invite new users, makers, and developers into the open-technology community, and increase the reach and capabilities of aquatic germplasm repositories

Low-Cost Resin 3-D Printing for Rapid Prototyping of Microdevices: Opportunities for Supporting Aquatic Germplasm Repositories

Germplasm repositories can benefit sustainable aquaculture by supporting genetic improvement, assisted reproduction, and management of valuable genetic resources. Lack of reliable quality management tools has impeded repository development in the past several decades. Microfabricated open-hardware devices have emerged as a new approach to assist repository development by providing standardized quality assessment capabilities to enable routine quality control. However, prototyping of microfabricated devices (microdevices) traditionally relies on photolithography techniques that are costly, time intensive, and accessible only through specialized engineering laboratories. Although resin 3-D printing has been introduced into the microfabrication domain, existing publications focus on customized or high-cost (>thousands of USD) printers. The goal of this report was to identify and call attention to the emerging opportunities to support innovation in microfabrication by use of low-cost (<USD 350) resin 3-D printing for rapid prototyping. We demonstrate that low-cost mask-based stereolithography (MSLA) 3-D printers with straightforward modifications can provide fabrication quality that approaches traditional photolithography techniques. For example, reliable feature sizes of 20 µm with dimensional discrepancy of <4% for lateral dimensions and <5% for vertical dimensions were fabricated with a consumer-level MSLA printers. In addition, alterations made to pre-processing, post-processing, and printer configuration steps improved print quality as demonstrated in objects with sharper edges and smoother surfaces. The prototyping time and cost of resin 3-D printing (3 h with USD 0.5/prototype) were considerably lower than those of traditional photolithography (5 d with USD 80/prototype). With the rapid advance of consumer-grade printers, resin 3-D printing can revolutionize rapid prototyping approaches for microdevices in the near future, facilitating participation in interdisciplinary development of innovative hardware to support germplasm repository development for aquatic species

Machine-learning-assisted spontaneous Raman spectroscopy classification and feature extraction for the diagnosis of human laryngeal cancer

Germplasm repositories can benefit sustainable aquaculture by supporting genetic improvement, assisted reproduction, and management of valuable genetic resources. Lack of reliable quality management tools has impeded repository development in the past several decades. Microfabricated open-hardware devices have emerged as a new approach to assist repository development by providing standardized quality assessment capabilities to enable routine quality control. However, prototyping of microfabricated devices (microdevices) traditionally relies on photolithography techniques that are costly, time intensive, and accessible only through specialized engineering laboratories. Although resin 3-D printing has been introduced into the microfabrication domain, existing publications focus on customized or high-cost (\u3ethousands of USD) printers. The goal of this report was to identify and call attention to the emerging opportunities to support innovation in microfabrication by use of low-cost

Successful cryopreservation of coral larvae using vitrification and laser warming

Abstract Climate change has increased the incidence of coral bleaching events, resulting in the loss of ecosystem function and biodiversity on reefs around the world. As reef degradation accelerates, the need for innovative restoration tools has become acute. Despite past successes with ultra-low temperature storage of coral sperm to conserve genetic diversity, cryopreservation of larvae has remained elusive due to their large volume, membrane complexity, and sensitivity to chilling injury. Here we show for the first time that coral larvae can survive cryopreservation and resume swimming after warming. Vitrification in a 3.5 M cryoprotectant solution (10% v/v propylene glycol, 5% v/v dimethyl sulfoxide, and 1 M trehalose in phosphate buffered saline) followed by warming at a rate of approximately 4,500,000 °C/min with an infrared laser resulted in up to 43% survival of Fungia scutaria larvae on day 2 post-fertilization. Surviving larvae swam and continued to develop for at least 12 hours after laser-warming. This technology will enable biobanking of coral larvae to secure biodiversity, and, if managed in a high-throughput manner where millions of larvae in a species are frozen at one time, could become an invaluable research and conservation tool to help restore and diversify wild reef habitats

Conduction‐Dominated Cryomesh for Organism Vitrification

Abstract Vitrification‐based cryopreservation is a promising approach to achieving long‐term storage of biological systems for maintaining biodiversity, healthcare, and sustainable food production. Using the “cryomesh” system achieves rapid cooling and rewarming of biomaterials, but further improvement in cooling rates is needed to increase biosystem viability and the ability to cryopreserve new biosystems. Improved cooling rates and viability are possible by enabling conductive cooling through cryomesh. Conduction‐dominated cryomesh improves cooling rates from twofold to tenfold (i.e., 0.24 to 1.2 × 105 °C min−1) in a variety of biosystems. Higher thermal conductivity, smaller mesh wire diameter and pore size, and minimizing the nitrogen vapor barrier (e.g., vertical plunging in liquid nitrogen) are key parameters to achieving improved vitrification. Conduction‐dominated cryomesh successfully vitrifies coral larvae, Drosophila embryos, and zebrafish embryos with improved outcomes. Not only a theoretical foundation for improved vitrification in µm to mm biosystems but also the capability to scale up for biorepositories and/or agricultural, aquaculture, or scientific use are demonstrated