Texas Tech University

NSRL Receives NSF Grant to Upgrade Genetic Resources Collection to Liquid Nitrogen Preservation

Background

The Genetic Resources Collection (GRC) of the NSRL currently (November 2021) contains more than 455,000 tissue samples from more than 100,000 individuals representing approximately 1,100 species of mammals and other taxa. It is among the largest, fastest growing, and most utilized collection of its kind. These samples are used by researchers from around the world to address questions vital to science and society, such as advancing our understanding of global biodiversity, the impact of humans and their activities on the natural world, and the transmission of disease between wildlife and humans. Each tissue sample is unique and irreplaceable, as it represents an individual at a specific point in time and space. The samples are of unlimited actual and potential value to the science community in terms of on-going and future research. It is crucial that these tissues be preserved at the highest standards to ensure their long-term availability for maximum scientific research potential and discovery.

Since its establishment in the 1970's, the GRC had relied upon traditional -80°C mechanical freezers for storage of the GRC's frozen samples. However, these freezers have a short lifespan and are subject to frequent malfunction or complete failure, as well as to power outages, thus putting samples at risk for degradation or total loss. More importantly, current and developing scientific disciplines require that tissue samples be preserved at temperatures below -132°C to preserve the entire spectrum of genomic data they contain (e.g., RNA, viruses, bacteria). Storage in liquid nitrogen (LN), which maintains samples near -190°C, thus has become the "gold standard" for preservation of tissue collections.

In 2015, Dr. Robert D. Bradley (Director of the NSRL) and Dr. Robert J. Baker (Former Director of the NSRL, deceased) received a grant from the Collections in Support of Biological Research program of the National Science Foundation (NSF) to transfer the frozen tissues of the GRC from the mechanical -80°C freezers to vapor-phase liquid nitrogen freezers in order to ensure the collection's long-term preservation and availability for scientific research.

The 3-year project included the transfer of nearly all frozen tissue vials to stainless steel boxes with polypropylene cell dividers. The process of transferring tissues to new boxes and installation in the LN freezers was time and labor intensive, but it allowed for confirmation and updating of the GRC's existing inventory of the tissue collection, as well as the upgrading of some vials with new label stock and barcodes (as needed). Dr. Caleb Phillips (Curator of the GRC) oversaw the project and ensured that the NSRL was incorporating the best museum practices in tissue archival and database methodologies as the transition took place.

Other outcomes of this project included: expanded storage capacity of the GRC, allowing for growth of the collection; annotation of the NSRL public database with genetic sequence database numbers; annotation of the NSRL's internal database to indicate samples obtained from virus-positive vouchers and designation of those samples with color-coded labels; labeling of symbiotype and holotype genetic samples with color-coded labels; education and training of graduate and undergraduate students in collection management practices; a public exhibit highlighting the significance of genetic resource collections to science and society; testing of cryolabels and other archival supplies; and reduced energy consumption by the transition to a green technology.

Progression of the Liquid Nitrogen Conversion Project

August - December 2015: The GRC facility was fully renovated to accommodate the LN system and to upgrade the overall facility. TTU provided the funding for the renovations of the GRC facility, as well as the purchase of two of the five LN freezers necessary to house the frozen tissues inventory and allow for growth of the collection.

January 2016: Five Taylor-WhartonTM LABS freezers, supplied with liquid nitrogen via an exterior bulk tank and vacuum-jacketed piping, were installed. Initially, three of the 5 freezers were filled with liquid nitrogen, with others filled and put to use as needed. With the assistance of Museum Science graduate students hired with NSF funds, the process of inventorying the collection, re-labeling vials (as needed), transferring samples to new boxes, and installing them into the LN freezers began.

June 2016: At this point, more than 20,000 samples were fully processed (sorted, labeled, inventoried, and cell boxed) and placed in the LN freezers. More than 50,000 additional vials were in various stages of processing. Student Assistant Taylor Soniat began a study to determine degradation of DNA in frozen liver samples collected over a 30-year period.

May 2017: More than 72,000 samples were fully processed and installed into the LN freezers. The entire Radioactive Collection of 13,922 samples was processed and installed into the 20K LN freezer designated exclusively for that collection. All holotype and symbiotype (host species of new virus strains) tissue samples were identified, consolidated in color-coded and labeled boxes, and housed in a designated rack and freezer for security purposes. Taylor Soniat completed his study of DNA quality and presented his results at the meetings of the Texas Society of Mammalogists and Society for the Preservation of Natural History Collections.

July 2018: By this date, a total of more than 229,000 samples were fully processed and installed into the five LN freezers. More than 54,000 additional samples were processed but required transfer to steel boxes and inventory updates before being transferred to LN. All known virus-positive samples were identified in the NSRL internal database and on tissue box labels. All GenBank accession numbers associated with GRC samples were downloaded from GenBank and entered into the NSRL's internal database.

May 2019: The NSF-funded project was completed. A total of 292,751 samples, comprised of those previously housed in -80C mechanical freezers as well as new samples accessioned over the course of the grant period, were installed in liquid nitrogen freezers. At that time, nearly all available LN freezer space was occupied (4 of 5 freezers were full), but space for ~12,000 samples had been reserved in the fifth freezer to allow for continued growth of the collection. Due to space restrictions, the decision was made to retain ~36,000 samples (the oldest samples in the GRC) in -80C freezers, but they were fully processed and ready to move to liquid nitrogen freezers when space becomes available. Piping was installed to accommodate one additional freezer when funds become available for that purchase, and the NSRL is actively seeking funding sources to further expand the LN system.

Over the course of the grant period, 9 graduate students (7 Museum Science and 2 Biological Science) received training and experience in the GRC while contributing a total of 8,643 hours of effort toward processing, inventorying, databasing, and installing samples.

Taylor Soniat, a Biological Sciences student and a GRC Student Assistant, utilized GRC samples for his Master's research project on degradation over time of DNA in samples held in -80 freezers. Taylor presented the results of his research at the annual meetings of the Texas Tech Biological Sciences symposium, the Texas Society of Mammalogists, and the Society for the Preservation of Natural History Collections. Taylor's thesis was titled "Assessing levels of DNA Degradation in Frozen Tissues Archived under Various Preservation Conditions in a Natural History Collection". As a result of his research and experience in the GRC, Taylor was the recipient of an ORISE (Oak Ridge Institute for Science and Education) Fellowship to work in the Biorepository of the U.S. Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia.

Heidi Stevens, a Museum Science student who was funded as a GRC student assistant on the NSF grant, conducted her Master's thesis research on the degradation rate of DNA that occurs after death of an organism. She graduated in August 2021, and her thesis was titled "Temporal Rate of Post-mortem DNA Degradation in Archived Samples: Evidence from Liver and Muscle Tissues". Heidi is now employed by the NSRL as a Research Aide.

A Museum exhibit funded in part by the NSF grant, entitled "Frozen in Time," was developed and open to the public October 2019 to January 2020. The exhibit featureed the history of the NSRL's GRC, including the NSF project, and highlighted the value of genetic resource collections to science and society.

Most significantly, this project provided for the long-term protection of the current and future samples in the GRC and ensures that the collection can supply the scientific community with the highest quality genetic materials for research.

For additional information or updates, contact Lisa Bradley, Research Associate, lisa.bradley@ttu.edu.

Natural Science Research Laboratory