Introduction

A key aspect of biological research revolves around the gathering and collection of samples and their preservation for examination and analysis at a future date. Since time elapses between when a sample is collected and when it is analyzed, and biological samples often degrade over time, it is imperative to have a process of storage (short and long term) that is efficient and preserves sample integrity over time. Today, billions of biological specimens and samples collected by researchers in academia, research institutes, hospitals and commercial organizations are often stored in cold environments (refrigeration @ ~40°C, low- and ultralow- temperature freezers at -50°C to -800°C, and liquid nitrogen @ -1700°C).

This article will highlight some of the shortcomings in the use of cold-temperature-based sample storage, describe the new and innovative technologies available today that mitigate these shortcomings, and offer suggestions on the convergence of these technologies in meeting the global challenge to be faced as bio-specimen collection increases in research labs as well as in bio-banks.

Current best practices of cold storage of biological samples

In the United States alone, there are more than 40,000 individual research laboratories located on university campuses that are advancing the field of biological and biomedical sciences. Researchers within these laboratories have assembled a very large collection of biological samples from clinical and field studies, some irreplaceable, all representing enormous scientific and financial value for the researcher and the organization (universities, research institutes, biotechnology/pharmaceutical companies, biobanks, etc.). The cost per sample collected can range from a few dollars up to $10,000.¹ There are currently over a billion samples (DNA, RNA, cells, clones, tissue organs, blood, buccal swabs, etc.) collected and warehoused in thousands of research labs and bio-banks globally. These samples are of high value to researchers, and current research trends are driving growth of these collections at an escalating rate.

To preserve these important research assets, organizations and individual researchers engaged in biological and biomedical research invest a huge sum of money in capital equipment purchases and maintenance of cold storage facilities to stabilize and store their large inventory of samples. However, there are increasing disadvantages to this method. For example, cold refrigeration/freezers produce hydrofluorocarbons, which are some of the most potent greenhouse gas pollutants² with a deleterious impact on the environment. Quantitatively, according to a report in The Economist, the typical ultra-low-temperature freezer consumes about 7,665 kWh per year while releasing 54,805 pounds of carbon dioxide. This is equal to the emission from about four cars.³

Additional challenges to the use of cold storage are highlighted below:

• Cold packing and shipping produce a large amount of waste materials.
• Purchase costs, maintenance costs and energy costs of cold refrigerators/freezers add up as sample collection grows, and accelerate as the cost of energy increases.
• Heat generated from refrigerators/freezers further adds demands to facility requirements, costs and planning to stabilize environmental conditions at lower temperatures than would be required without the equipment.
• Freezers take up an increasingly large amount of lab space, potentially inhibiting current and new facility/ research space needs.
• Multiple freeze-thaw cycles can lead to sample quality degradation.
• Cold freezing, especially of bio-tissues, can lead to cell membrane damage.3
• Power failure or freezer failure can place samples at risk for degradation and loss.

New approaches to bio-sample storage and archiving

In recent years, new technologies developed for the stabilization and storage of biological samples at room temperature have garnered a lot of attention.⁴ While these technologies differ in their implementation, the overall paradigm remains the same—provide room temperature stabilization, storage and archiving of biological samples. A leading innovator in room temperature bio-sample storage is San Diego, California–based Biomatrica Inc.

Biomatrica’s room-temperature sample stabilization and storage technology is based on extremophile biology that allows some organisms to survive in a dry state (anhydrobiosis) for >100 years.⁵ Anhydrobiotic organisms can protect their DNA, RNA, proteins, membranes and cells for long-term survival in a dried state and later be revived by simple rehydration. The method transfers the natural molecular principles of anhydrobiosis to a synthetic chemistry–based stabilization science that works by forming a thermo-stable barrier around the sample, protecting it from degradation during storage at room temperature (see Figure 1). Sample recovery is as simple as rehydrating the mixture, making the sample ready for use in a wide range of downstream applications such as microarray analysis, DNA/RNA sequencing, end-point PCR, cDNA synthesis, reverse transcription, etc.

Figure 1. A molecular-level view of the process of sample preservation by Biomatrica’s SampleMatrix (a) by mimicking the process of anhydrobiosis (b).

Biomatrica

The ability to stabilize, transport, store and archive biological samples at ambient conditions now offers possibilities of increased sustainability for managing biological research in the life science industry. A recent collaboration between the Sorenson Molecular Genealogy Foundation (SMGF) and Biomatrica involved the application of Biomatrica’s SampleMatrix® roomtemperature storage technology to archive SMGF’s current DNA samples and the long-term storage of all newly collected samples.⁶ In addition, SMGF plans to move its collection of previously archived samples from cold-temperature freezers to room-temperature storage. According to Scott Woodward, executive director of SMGF, “Biomatrica has developed a product that we feel addresses our concerns in a very practical, economical and secure way.”

Are the benefits to room-temperature storage achievable?

A number of advantages have been ascribed to room-temperature sample storage over the current cold-temperature-based storage. Perhaps the most significant benefit is in the area of increased sustainability of biological research when samples are transitioned from cold freezers to ambient room temperature. This process should lower energy consumption, reduce the carbon footprint (CO2 emission), lower the costs associated with equipment purchase and maintenance, lower material costs, and provide better utilization and optimization of lab space gained by retiring the cold freezers.

In order to demonstrate the associated benefits of the transition from cold-temperature sample storage to room-temperature storage, a university in California conducted a pilot study as part of a campus-wide energy sustainability initiative. The study involved 12 laboratories within the university, representing a broad range of research areas and lab sizes, and about 60,000 samples out of the nearly 1 million identified as transferable from cold freezers to room-temperature storage.⁷

The major goal of the study was to better understand the financial and environmental cost savings associated with a move away from cold storage to ambient room-temperature technology. A sophisticated forecast model was developed using information from the pilot group, data from the university, and industry trends in order to estimate the potential campus-wide benefits to increased sustainability via the adoption of this technology.

Figure 2. Projected savings in energy (BTU) and cost ($) over 10 years by switching from cold-freezer storage samples to room-temperature storage (data from Ref 7).

Stanford University

The final report provided 10-year projections for maintaining the current cold storage freezers at ~564,000 MBTU of energy consumption at an energy cost of ~$70 million, while generating >50,000 metric tons of CO2 emissions.⁶ These findings are clear demonstrations of an unsustainable path with current storage systems, especially when the industry projects a significant increase in the collection of bio-specimen samples. In contrast, over the same 10-year time frame, the study projects (see Figure 2) that transitioning from cold storage to room-temperature storage solutions for all identified samples in the university laboratory freezers would generate cost savings between $11 million and $20 million and energy savings of ~160,000 MBTU, prevent 17,000–20,000 tons of greenhouse pollutant (CO2) from entering the environment, and recoup the initial storage transfer costs within five years.⁷

Studies such as those described here clearly suggest a robust solution to sample storage, with significant and measurable benefits to adoption of this technology. The increasing diversity and size of bio-specimens routinely collected will require a highly scalable, cost-effective and environmentally friendly storage, stabilization, transport and archival system such as is offered by room-temperature sample storage technologies.

Conclusions

Can biological samples be safely stored at room temperature for long periods without the risk of sample degradation? That is a question that has been easily answered by the new technologies emerging from such companies as Biomatrica and amply demonstrated from

As sample collection grows as anticipated for a wide range of biological and biomedical research, storage and archiving of these important research assets will be critical. Will the future of sample storage remain at the status quo, i.e., cold storage, or will the clear advantages offered by new technologies such as room-temperature storage and archiving assume a more central role? The energy sustainability pilot study and its findings clearly demonstrate significant benefits in adopting room-temperature storage solutions as a viable alternative to or replacement for cold-storage systems.

In summary, ambient room-temperature storage offers an eco-friendly and highly sustainable process to manage biological samples, enhance lab space conservation via freezer retirements, provide a more efficient and cost-effective transportation of samples, and reduce the risk to sample loss from vulnerable freezer-dependent issues such as fire, temperature fluctuations or other acts of nature.

References

1. Iyengar, G.V., Subramanian, K.S., Woittiez, J.R.W., Element Analysis of Biological Samples: Principle and Practices: http://books.google.com/books?id=l5bcrKai_7kC&pg=PA82&lpg=PA82&dq=freezing+of+biological+samples&source=bl&ots=dc78yT6qHC&sig=8s6P4fB Tr6Jdvap-nRlDsQOfGQw&hl=en&ei=042FSuumKGTtgfu4OSvCg& sa=X&oi=book_result&ct=result&resnum=5#v=onepage&q=&f=false

2. "Trading Thin Air," The Economist, published May 31, 2007: https://www.economist.com/special-report/2007/06/02/trading-thin-air

3. Stouten, C.W., Cunningham, M. Freezing Biological Samples: http://www.myneurolab.com/global/Manuals/Tips%20and%20Techniques%20Freezing%20Artifact.pdf

4. (a) Hernandez, G.E., Mondala, T.S., Head, S.R., Assessing a Novel Room-Temperature RNA Storage Medium for Compatibility in Microarray Gene Expression Analysis, BioTechniques, August 2009; 47: 667-670 http://www.biotechniques.com/multimedia/archive/00052/ BTN_A_000113209_O_52922a.pdf;
(b) Ohgi, S., Making RNA More Durable at Room Temperature: http://www.genengnews.com/articles/chitem. aspx?aid=2241;
(c) No Fridge Required: https://www.economist.com/technology-quarterly/2007/12/08/no-fridge-required;
(d) Protecting RNA Samples at Room Temperature: https://www.labmanager.com/how-it-works/2008/07/protecting-rna-samples-at-room-temperature

 5. Crowe, J.H., Carpenter, J.F., Crowe, L.M. The Role of Vitrification in Anhydrobiosis, Ann. Rev. Physiol. 1998, 60: 73.

6. http://www.biomatrica.com/media/pr/0006-sorenson.pdf

7. Jensen, G., Room Temperature Biological Sample Storage; May 2009: http://sustainablestanford.stanford.edu/sites/sem. stanford.edu/files/documents/Stanford_Room_Temp_Pilot_ May09.pdf