The Process Is the Product:How Cryopreservation Protocols Impact Sample QualityBy Allison Hubel, Ph.D., University of Minnesota

The material presented in this article was taken from a scientific symposium held at Genentech in February of 2016. The symposium was sponsored by Brooks Life Science Systems and focused on high quality sample management with specific focus on the process and protocols.Material in this article is taken from a presentation made by Allison Hubel, PhD of the University of Minnesota, http://www.biocor.umn.edu/. Dr. Hubel was joined by other co-presenters to discuss global sample management standards and improving sample quality input for research.

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The Process Is the Product: How Cryopreservation Protocols Impact Sample Quality

By Allison Hubel, Ph.D., University of Minnesota

The material presented in this article was taken from a scientific symposium held at Genentech in

February of 2016. The symposium was sponsored by Brooks Life Science Systems and focused on high

quality sample management with specific focus on the process and protocols.

Material in this article is taken from a presentation made by Allison Hubel, PhD of the University of

Minnesota, http://www.biocor.umn.edu/. Dr. Hubel was joined by other co-presenters to discuss global

sample management standards and improving sample quality input for research.

Introduction

The demand for biospecimens – macromolecules, cells, or tissues – is growing rapidly. Therapeutic need

is surging as people are treated with cells or tissues to cure a wide variety of diseases. Plus, entirely new

therapies are being developed for long standing health concerns, such as cancer. Biospecimens are also

being used for diagnostic and epidemiologic purposes -- to diagnose diseases, improve blood safety, and

monitor treatment progression.

Most biospecimens are collected in one location at a given time, and stored and used at another

location at a later time. A blood specimen taken at the doctor’s office may be shipped off and analyzed

at a variety of different places. And that healthcare provider is collecting more specimens than ever

before. The critical biological properties of that sample must be preserved, or the measurement will not

be clinically valid.

The preservation of those samples must be built on scientific principles and protocols. Cryopreservation

is a process framed by those scientific principles and not a cold black box. When a sample is placed in a

freezer or dewar, it is not magically preserved in pristine condition. Understanding the scientific

underpinnings of cryopreservation is critical to understanding and maintaining high-quality

biospecimens for whatever application.

Why are samples frozen?

There are two reasons why samples are frozen. First, there is liquid water in biospecimens that

participates in chemical reactions that degrade the biological entity whether it’s a molecule, cell, or

tissue. When frozen, the mobility of the water molecules present in the sample is greatly reduced.

Second, every biospecimen contains molecules that can act to degrade its biological system, such as

enzymes. When the temperature is reduced, the degradative activity slows down, helping to stabilize

the system.

Components of a Cryo Preservation Protocol

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The development of an effective preservation protocol requires avoiding a series of seemingly subtle

mistakes, each of which can result in reduced post-thaw quality and poor outcomes. A preservation

protocol typically consists of a standard set of components, whether involving a molecule, cell, or tissue

(figure 1).

Pre-analytical Phase

With biofluid samples, the time delay between collection and processing, the temperature at which the

sample is held, and delays in centrifugation are core parameters that can affect sample quality. In

addition, residual drugs, anesthetics, and anti-coagulants may be present in the biospecimen and

influence quality both pre-freeze and post-thaw. An NIH study drew whole blood from a donor and

analyzed two different vials stored at room temperature for 2 hours. Researchers found 35 different

proteins that were statistically significant between the two samples. Protease inhibitors had little effect

and the effects were cell mediated. Cells stressed by the lack of oxygen and low temperatures produced

proteins that had a profound influence on the composition of the sample.

With tissue biospecimens, it is known that the type of collection, whether a biopsy or autopsy, is

important. Is the ischemia cold or warm? What was the duration of those ischemia times? What were

the fixation method and time? These factors all can impact sample quality.

Another study using gene arrays on colon cancer biospecimens showed that within 15 minutes after

clamping the blood supply from the tumor, there was a change in 10% to 15% of the genes. Within 30

minutes, 20% of the genes had changed. As a result, the study recommended that the tumor tissue be

placed in liquid nitrogen within 30 minutes of the clamping of the blood supply.1

We know that cells respond to their environment. When removed from the body, cells experience

hypoxia and hypothermia. These are well-understood pathways of stress response that involve secretion

of molecules. There is degradation of RNA and cell death. These cell stress pathways are well studied

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and are relevant for both the storage of biospecimens plus understanding stroke and organ

preservation.

It is critical to record the preanalytical phase of a biospecimen. There is standardized labeling — called

standard pre-analytical coding for biospecimens (SPREC). This is a published standard that was produced

by ISBER in 2012. If pre-analytical variables cannot be controlled, they must be notated for reference at

a later time. Even if samples are collected at clinical sites that are not at your location, their pre-

analytical variables can be noted to ensure biospecimens are properly annotated and end users know

the manner by which the samples have been processed.

Introduction of a Cryopreservation Solution

Cells can’t be removed from the body, stored in a freezer, and be expected to survive. Certain reagents

must be added to the cells to improve their ability to endure freezing and thawing. In the 1970s,

dimethlysulphoxide (DMSO) was identified as an effective additive for preserving cells. It is one of many

classes of molecules that can act to stabilize the cell during the freezing process.

Stabilization of proteins has been studied for many decades and in many situations. Retaining their

function requires the use of stabilizing agents — the most common being sugars. Studies have also

recommended the use of stabilizing agents in serum and plasma.

Freezing Protocol

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Since the 1970s, it’s been known that the rate (C/min) at which a cell freezes has a strong influence on

its ability to survive freezing and thawing. Figure 2 illustrates the elements of a controlled-rate freezing

protocol: initial equilibration phase, initial cooling, seeding step, secondary cooling, and cooling to final

temperature. The goal is to carefully control the temperature versus time that the sample is traversing

from 4° C to liquid nitrogen temperatures.

When samples are placed in a -80° C freezer and passively frozen, the average cooling rate is between 7°

C per minute and 1° C per minute. Cooling at the slower rate requires adding insulation to the sample to

slow cooling. It’s important to know that the cooling rate decreases as the sample cools. The actual

cooling rate will also vary with the load (number of bags/vials) in the storage unit.

Pure water melts at 0° C. However, it does not freeze at that temperature. In a practical situation, a 1 ml

vial of water will start forming ice at -7° C to -9° C. If solutes are added, it will depress the freezing

temperature. There is no other molecule that undergoes a phase transition from a liquid to a solid that

undercools as much as water. And this attribute makes reproducibility of the freezing process a

significant challenge.

Frequently, solids and liquids reside together at the same time in these complex solutions. The solute is

not incorporated in the ice, so there is a partitioning of what is freezing, plus liquids and solids are

freezing over a range of temperatures.

When viewing low-temperature RAMAN microspectroscopic representations of frozen samples, the

black regions are ice and the green areas are the unfrozen fractions adjacent to the ice crystals (figure

3). The illustration in white represents a 1 micron gap between ice crystals. In this gap, the interaction

between the ice and the liquid resulted in the aggregation of the proteins and denaturation. As the

temperature decreases, the gap between adjacent ice crystals freezes and the cells are squeezed into

that region. The concentration in the gap increases with decreasing temperature. This means that the

chemical and mechanical environment for the biospecimen is changing as the freezing occurs.

Storage Conditions

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There are a variety of options for storage — mechanical freezers and liquid nitrogen dewars to name a

few. And these storage systems operate from ambient to -190° C. Today, there are millions of

biospecimens stored at –20° C.

As described previously, we freeze biosamples to slow down the molecular mobility of the water

molecules and the activity of degradative molecules. Freezing ends at two points: either the eutectic

temperature (-21° C for isotonic saline) or the glass transition temperature (-132° C for pure water).

There are phase diagrams that can be used to understand the end of freezing — the full solidification of

the water molecule.

Within the plasma proteome, there are about 15,000 proteins and their concentration varies over about

15 orders of magnitude (figure 4). Hundreds of those proteins can degrade other molecules, and they

are present in all samples.

Protein activity is a function of its molecular dynamics. Reducing the temperature reduces the activity of

the molecule and the Arrhenius Equation (figure 5) can help us understand the dependence of that

relationship with temperature. There is a threshold temperature below which no activity is observed.

Enzymatic activity can also be significant down to very low temperatures — -80° C or lower. Certain

RNAses have been observed to be active at temperatures approaching -93° C. This is another reason

why RNA is not stable when it is stored at -80° C.

Conventional wisdom dictated that the magic temperature at which degradative molecular activity

stopped was -53° C. Therefore, samples stored at -80° C should be fine. Unfortunately, the degradative

effects of a variety of molecules have been measured down to lower temperatures. Samples must be

stored at temperatures at which they solidify and below the temperature at which all of the degradative

molecules are inactivated.

Warming Protocol

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The sample has been stored and now it’s needed. After shipment to the point of use, it must be

warmed. The same range of temperatures must be traversed during warming as were traversed during

freezing. Recrystallization damage and long-term exposure to high concentrations can occur, which can

be damaging to proteins, cells, and tissues.

Rapid warming (80° C per minute) was found to be helpful for conventional thawing of cells.2 With

proteins, the same was found true. However, slow warming (0.8° C per minute) was damaging to cells.

For example, when observing a 1 ml vial in a 37° C water bath, there was rapid warming when the

sample was very cold (figure 6). As the sample warmed up, the temperature change reached a plateau

as the melting process began. The warming was being converted to a change in phase, causing the

sample temperature to remain constant. This is an example of the proper technique for sample

warming.

Post-Thaw Assessment

Post-thaw assessment is very important. Studies suggest that the methods of sample preparation will

influence the outcome. Proteomic analysis is influenced by vortexing, the use of protease inhibitor, the

selection of the sample container, and any alterations in post-thaw pH. These factors all have the

potential to influence the downstream use of that biospecimen. A personal anecdote illustrates this

issue.

At a cell therapy clinical trial in the 1990s, we were ready to start patient treatment. We produced 12

different aliquots of the lymphocyte. The first bag was thawed out, placed in the bioreactor to expand it,

and then the cells were transfused with the corrective gene. The test failed. Another bag was thawed

out and the same thing happened. The third aliquot was put into a bag and the cell count was

measured. We observed that the cell count dropped precipitously over time, post thaw (figure 7). As a

result, a pre-culture step was instituted with FDA approval before we inoculated into the bioreactor. It

was clear the failed outcomes resulted from post-thaw apoptosis of the cells.

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This example demonstrates that the timing of the assessment of the cells, post thaw, is critical. Cell

viability and recovery may be different depending upon the time post thaw that the assay is performed.

It is important to perform post-thaw assessment at the same time.

If a cryopreservation protocol is a process, and you stop the process at any given point to do process

monitoring, you can detect the point at which the process has failed. Metrics must be established to

measure the quality of the sample. For example, these might be cell recovery time or the stability of a

specific biomarker. Process monitoring should happen at different points in protocol development

Container selection is another factor that impacts post-thaw assessment. Straws, vials, and bags must be

designed for cryogenic environments to be effective. The wrong container can mean the loss or

contamination of a sample.

In most situations, biosamples cannot be repurified or reprocessed, post thaw. In cells, cryopreservation

agents cannot be washed out or completely removed. Samples must achieve USP quality.

Summary

The cryopreservation process is the product. What you do along this path will have a significant

influence on sample quality. Understanding the process and the scientific principles behind them will

help identify where things go wrong and develop rational strategies to solve problems.

References

1 Spruessel, BioTechniques, 36:1030, 2004

2 Cao, Biotech. Bioengr 2002

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About the Author

Dr. Allison Hubel is professor of mechanical engineering at the University of Minnesota, and is director

of the Biopreservation Core Resource (BioCoR), the nation’s only resource on biopreservation. The

mission BioCoR is to advance the science, technology and practice of preservation. BioCoR offers two

short courses: Preservation of Molecular, Cellular, and Tissue Biospecimens and Preservation of cellular

therapies. More information on these courses can be found on the BioCoR website

(www.biocor.umn.edu). Information on best practices in preservation can be found in the BioCoR

library, and there is a monthly newsletter for people interested in understanding best practices for

preservation. Dr. Hubel has published many scientific articles on preservation and was recently deputy

editor of Biopreservation and Biobanking, the official journal of the International Society for Biological

and Environmental Repositories (ISBER).