The basics of cryopreservation

By Carol Horton

Hand holding an ATCC Mini vial taking a sampleAt the heart of ATCC biobanking is the seed stock concept—this is similar to what you would call your master cell and working cell banks. We utilize this methodology to keep all distribution lots as close to the original depositor’s material as possible; this includes consideration of not only passage and purity, but also phenotypic and genotypic characteristics of individual strains. By performing analyses on cultures at each step (from initial deposit to seed stock to distribution stock), we generate a three-fold data set comparing the stability of the culture’s attributes over passage and preservation.

Preservation becomes essential in the routine maintenance of cultures, allowing us to minimize passage in vitro and preserving valuable stocks used or developed in research. The most common methods of preservation include cryopreservation, which is the process of freezing cultures, and lyophilization, which is the process of freeze-drying material for longer-term storage and stability. Central to both techniques is the management of water during the preservation process. Here, we will discuss the basics of cryopreservation.

When an aqueous suspension of living cells is frozen, ice forms first in the solution around the cells. This, in turn, increases the concentration of solutes in the local environment. As a result of this change in osmotic pressure, water migrates from the cells as long as the imbalance of salts remains outside of the cells. The amount of ice that forms in the solution, and the subsequent dehydration of the cells, is dependent upon the rate of cooling and cell permeability. In fact, there is a delicate balance in water and solute management during the preservation process. If too much water migrates out of the cells, the internal salt concentration rises quickly, which could cause cell damage, leading to poor post-freeze viability, or even to cell death. In contrast, if too much water remains inside the cells, then a greater number of ice crystals will form, which could cause internal damage or complete cell lysis upon thaw.

So, how do we manipulate the environment to minimize cell damage during the preservation process? For starters, we add a cryoprotectant, such as glycerol or DMSO, which either binds salts or alters cell permeability while decreasing the rate of cellular dehydration. We also control the rate of cooling, keeping it at a steady -1°C/minute until the culture reaches a stable frozen temperature of -70°C to -80°C. After the culture has reached a stable frozen temperature of -70°C to -80°C, the optimal next step for long-term storage would be to move the culture to the vapor phase of liquid nitrogen. This ultra-low temperature is often used for periods longer 5 years, since ice crystals continue to form in the frozen suspension when stored at temperatures above -130°C. However, most bacteria and sporulating fungi can survive storage at -70°C to -80°C for 5 years or less; the actual length of time an individual strain can be stored at this temperature should be evaluated by performing yearly viability testing.

When recovering cryopreserved cultures, thawing should be rapid as this will minimize the damage from ice crystals that have formed inside the cells. Further, to optimize post-freeze growth, ATCC also recommends that cultures be initiated in the same medium and atmospheric conditions they were grown in prior to freezing.