Selecting the optimal storage temperature for your biospecimen (Part 1)
The time and distance between sample procurement (whether it is in a hospital, clinic, laboratory, battlefield or home) and biospecimen utilisation can range from minutes to years. This makes the quality of sample integrity extremely important in order to compare various studies over time.
As long as the sample is frozen does it matter what temperature it is stored at?
Many researchers will freeze samples according to what has been done before but there is a science to this process that many people may not have thought about.
Prof. Allison Hubel from University of Minnesota, US has worked on understanding this process for many years. Freezing changes the molecular mobility of a sample; in the case of water changing from liquid to solid form freezing will reduce its mobility and in the case of enzyme activity a reduction in reaction/activity rates will be experienced.
“If you have ever eaten an ice pop (popsicle) then you will understand the science of freezing” states Prof. Hubel
When you suck on an ice pop you find the colour disappears and you are left with clear ice. This is because complex mixtures freeze over a range of temperatures and you can be left with solids and liquids together in a frozen sample although to the eye the whole sample appears frozen. In the case of the ice pop, the coloured solute is not frozen but the water is so as you suck the liquid solute is sucked out first.
Prof. Hubel et al have recently reviewed in Biopreservation and Biobanking (BIO), 2014 the physical and enzymatic properties concerned with freezing to help you decide on the optimal temperature for storage of your biospecimen.
The physics of freezing
The thermodynamic state of multicomponent solutions can be described by phase diagrams and by time-dependent temperature-time-transition (TTT) diagrams. Specifically, the fraction of water that has been solidified and the corresponding unfrozen components can be determined by temperature. The onset of freezing is called nucleation and during this process water is removed from a sample in the form of ice, partitioning the sample and leaving unfrozen solutes behind. As the temperature decreases more water is removed and the solute concentration increases until the entire sample fully solidifies (at the eutectic temperature, Figure 1a and b) or vitrifies (the glass transition temperature, Tg). When a cryoprotectant is added to cells, ice crystals are avoided and along with a slow freezing process, solutes or cells are vitrified in a solid state without any dehydration or disruption.
Figure 1: How water freezes: (A) water molecules (blue circles) form ice crystals and other components in the sample (green and red circles) are concentrated into gaps between crystals which leads to dehydration and damage to cells (B) (green shapes – cells, blue stars – ice crystals).
For each biospecimen the glass transition temperature is important and can be calculated using technical empirical formulas. This transition temperature is dependent on several factors including concentration, storage, temperature and time of storage.
The relationship between biological activity and temperature
All biological specimens contain degradative molecules. Lipases, carbohydrases, proteases and nucleases may be present in fluid and/or tissue biospecimens. Temperature has a strong influence on protein dynamics: lower the temperature, lower the activity and greater the stability of the sample, e.g. Rasmussen et al observed lack of RNase A binding to substrate at a temperature of -58oC (Nature 1992), however RNase A activity has been measured at -93oC (Tilton et al, Biochemistry 1992).
How to decide on the optimal temperature?
To avoid degradation of your biospecimen, measurement of water mobility and protein activity for each sample type would be highly beneficial however is not very practical in most situations.
Prof. Hubel suggests three options:
- Estimate glass transition temperature mathematically for each biospecimen
- Check scientific literature for stability studies (see Hubel et al, BIO, 2014)
- Use a quality control (QC/QA) program to determine stability for a sample and permit continuous improvement in storage practices
Now that you have chosen the optimal temperature for your sample collection the next step is to decide on how you can reliably maintain this temperature. Part 2 of this blog series will discuss issues that need to be considered when choosing your storage vessel of choice.
Prof. Hubel, Ph.D, M.S, B.S. Mechanical Engineering heads a group focused on biopreservation of biospecimens at the University of Minnesota, US. The usefulness of a biospecimen is determined in large part on our ability to preserve and retain its critical biological properties. Allison Hubel’s group are working on understanding the molecular mechanisms of damage during the freezing process and developing new devices to introduce and remove preservation solutions from cells http://www.me.umn.edu/people/hubel.shtml.
Prof. Hubel (far right) and her group at the University of Minnesota.