Freezing Protocols, Storage & Strategy

A key aspect of biological research concerns the gathering and collection of samples and their preservation for examination and analysis at a future date. Since time elapses between sample collection and analysis and biological samples often degrade over time, it is imperative to have a process of creating a stasis stage within the sample (post-processing cooling and solidification) and subsequently to provide a storage environment (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 @ -50°C to -80°C)

It is important to understand and highlight some of the shortcomings in the use of cold-temperature-based sample storage, and to review 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.

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. 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 the 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, mechanical refrigeration/freezers produce hydrofluorocarbons, which are some of the most potent greenhouse gas pollutants 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.

Redefining cryopreservation protocols, strategies and practices to be “fit for purpose” in reference to program goals will increase long term sample utility and value, protect the institutional investment in product, therapeutic material, and research while increasing efficiency and reducing costs and environmental impact

Additional challenges to the use of cold storage:

• Purchase costs, maintenance costs, and energy costs of mechanical refrigerators/freezers add up as sample collection grow and accelerate as the cost of energy increases.
• The 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.
• Power failure or freezer failure can place samples at risk for degradation and loss.

Scientists and Researchers choose cryopreservation to truly preserve the cells or tissues they are storing.  In most cases, preservation doesn’t simply enable the ability to measure a biomarker or analyte post-thaw, but provides for the living cell to be frozen, remain completely static and unchanged, thawed, and be fully functional and viable again.  The cells must be able to thrive, post-thaw.  To achieve this there are many important facets of cryopreservation such as the selection and addition of cryoprotective agents, freezing rates, nucleation, storage, controlled thawing, and washing.  The longest step, and often the most uncontrolled, is storage.  Here cells are placed into a cryo environment for months or years at a time.  The intent is to keep them safely frozen below the glass transition temperature (Tg) of water, -135C.  High-efficiency LN2 vapor freezers will easily keep sub -150C temperatures, but what happens during transient warming events?  What happens when racks, cryoboxes, and vials are removed temporarily during routine storage and access activities?  Are they in danger of warming above -135C and experiencing micro-thawing events?  We know from our experiments that 2mL samples warm at 1.2C/second when removed from an LN2 freezer.  This means an innocent 2mL vial removed from a -190C LN2 freezer can cross Tg in less than 50 seconds.  Yes, samples are at risk of warming above Tg during any exposures.  The way to prevent excessive warming is by understanding warming rates, exposure times, and then controlling and monitoring all user interactions.