Hamed Shamkhalichenar, Jin-Woo Choi and Terrence R. Tiersch
Oysters and fish represent the vibrant heritage of the Gulf Coast and a traditional way of life for the people of Louisiana. It is important to keep oyster and fish populations healthy and growing in the estuaries and along the Gulf Coast. The genetic resources of these fish and shellfish are what makes them suited for life here in Louisiana, and those genetic resources need to be protected.
Germplasm repositories for fish and shellfish preserve sperm, eggs or embryos and vital information about the species. These repositories are one of the best ways to protect genetic resources of aquatic species, including support of fisheries; improvement of aquaculture, which is also known as fish farming; and conservation of endangered species. Cryopreservation is a vital technique to protect genetic material by freezing at ultra-low temperatures. However, the survival rate of the genetic material depends on processing, freezing and thawing conditions. As a result, numerous specific protocols have been reported for cryopreservation of aquatic species because of their broad biodiversity. Unfortunately, there is almost no standardization in these protocols, and it is very difficult to repeat or evaluate them. This has prevented the large-scale development of repositories for fish and shellfish and leaves them unprotected.
Also, research equipment, such as programmable freezers, can cost tens of thousands of dollars and are too bulky to be used in the field, greatly limiting the adoption of cryopreservation by many user groups. One way to overcome these barriers is to use electrical engineering to provide technology-based solutions for improvement of reliability and accessibility of cryopreservation. Monitoring other factors in addition to temperature during cryopreservation can provide additional information that can be used to assist a comparison of protocols and help us gain a better understanding of the complex dynamics of cryopreservation.
The Aquatic Germplasm and Genetic Resources Center of the LSU AgCenter was created in 2015 at the location of the former LSU Dairy Improvement Center to address the problems of repository development for aquatic species through interdisciplinary collaboration, such as between engineers and biologists. We have developed customized sensors to monitor the changes in temperature and electrical properties of samples during cryopreservation. When combined with a user interface and data storage and transfer capabilities, these systems can provide valuable information about freezing and thawing that can be used by the cryopreservation community to improve efficiency and reliability.
If we want these sensors to be widely used, they should be adaptable to a variety of user requirements. For example, different containers are used for freezing of samples ranging from straws to tubes and glass vials, and our sensors should be compatible with all of these. To do this we have incorporated fabrication methods, such as 3D printing and printed circuit board technology, to make customized sensors based on user needs. At present, consumer-level 3D printers are widely available across the world at a low cost. The instruction files used by these printers can be shared over the internet, and different user groups can use them to print sensors or modify them based on their requirements. Likewise, printed circuit board sensors can be fabricated or modified using design files through widely available manufacturing services. Because this technology is widely used to fabricate electronic components, the presence of many competitive fabrication services has reduced its cost drastically. This process of sharing design files across user communities is called open technology, and a great example is the sharing of files used to 3D print personal protective equipment, such as face shields, during the early stages of the COVID-19 pandemic.
In addition to sensing, we have developed low-cost portable monitoring systems to perform measurements and record and transfer data for storage. For example, we designed electronic circuitry to perform simultaneous precision measurements of temperature and electrical properties during the freezing process. In addition to the data from the sensors, these systems enable users to input and store information through interactive interfaces, such as LCD touch screens. Most importantly, we can include Wi-Fi and Bluetooth capabilities for data transfer to cellphones, laptops or cloud-based databases. This assists data management, such as quality control and quality assurance, which are essential for reliable handling of large numbers of samples in repositories.
To make sure these devices are widely accessible to the repository community, we use open-fabrication methodology in the design, prototyping and fabrication stages as we move through our alpha and beta testing process. In addition to our custom-made electronic boards, we also provide design files and fabrication instructions based on Raspberry Pi microprocessor boards and Arduino microcontroller boards and other off-the-shelf modules. These are inexpensive, consumer-level products that are widely available but are very powerful. This allows users with minimal experience in electronic systems to replicate devices with instructions or after watching YouTube tutorials. More advanced users can modify the devices to simplify designs or add more capabilities.
We are using this combination of sensors, monitoring and data management systems to facilitate standardization of cryopreservation in aquatic species. This will greatly assist protection of genetic resources of fish and shellfish, such as Louisiana oysters, in germplasm repositories. In addition, using these systems with our custom freezing devices will greatly reduce equipment costs and allow work in the field, which will expand the availability of cryopreservation and production of high-quality samples for repositories.
Hamed Shamkhalichenar was a postdoctoral researcher at the LSU AgCenter Aquatic Germplasm and Genetic Resources Center and is now an electrical engineer at Casana, a health care and technology company in Rochester, New York. Terrence R. Tiersch is a professor in the LSU AgCenter School of Renewable Natural Resources and is the director of the Aquatic Germplasm and Genetic Resources Center. Jin-Woo Choi is a professor in the LSU School of Electrical Engineering and Computer Science.
(This article appears in the summer 2021 issue of Louisiana Agriculture.)
Yue Liu, postdoctoral research associate at the Department of Biological and Agricultural Engineering and the Aquatic Germplasm and Genetic Resources Center, is inserting a sensor, fabricated by use of a printed circuit board, into a standard cryopreservation straw. A customized system with a user interface (touch screen) and data storage and transfer capabilities can provide valuable information about freezing and thawing that can be used by the cryopreservation community to improve efficiency and reliability. Photo by Olivia McClure
Researchers from the Aquatic Germplasm and Genetic Resources Center developed customized sensors to monitor the changes in temperature and electrical properties of sperm samples during cryopreservation. The figure shows sensors fabricated by use of printed circuit board being inserted into a standard cryopreservation straw and vial. Photo by Olivia McClure
A design diagram (upper panel) and actual (lower panel) 3D printed sensors used to measure temperature and electrical properties of samples during cryopreservation.
Novel sensors for measuring electrical properties of samples during cryopreservation have been custom designed using printed circuit board technology, which makes them cheap and widely available. Photo by Jonathon Lai
The sensors are part of custom-made monitoring systems. Devices such as these combine 3-D printing and custom printed circuitry with widely available off-the-shelf components. These devices are much cheaper and more flexible than commercially available instruments, are custom-designed for work with species such as oysters, can record data, and users can input sample information through a touch screen. The data can be transferred to a cellphone, laptop or a database using Wi-Fi or Bluetooth. Design files for these devices can be easily shared on the internet as open hardware to greatly expand their use to assist protection of genetic resources in repositories. Photo by Jonathon Lai