Friday, June 10, 2016

Lab Series# 12: Cryo-Preservation of cells


In an earlier post on PBMC isolation technique, I had talked briefly mentioned about cryopreserving cells for downstream processing. I kept the explanations in brief since the focus was on how to get a PBMC from whole blood samples. In this post, I will focus on details of cryopreservation of cells.

Cells have a basic metabolism requirement for survival and hence, all alive cells are metabolically active (By definition). However, when outside the body cells will exhaust their metabolic capability in relatively small time unless continuously maintained in a culture system. Practically a difficult process and then there is a problem of deviation from original properties of the cell to be studied. Cryopreservation provides a suitable alternative by storing the cells in revivable format. The very low temperatures reduce the metabolism rate to near zero.

Cryopreservation is defined as "Use of very low temperatures to preserve structurally intact living cells and tissues". There are several methods defined in the literature, describing the process of cooling the cells. The most well known among them is "Slow cooling technique" and second is a rapid method called "Vitrification". Vitrification is a cryopreservation technique that leads to a glass-like solidification of cells. Oocyte, zygote, embryo and blastocyst freezing by vitrification method for cryopreservation have been used for many years beside sperms preservation. Here I'm restricting the descriptions to slow cooling method. 

Table 1: Comparison of ultra-low temperature storage
methods for cell lines. Source
The process of cooling by itself can be lethal to the cells. There are arguments over what exactly causes cell damage. One theory suggests that the slow freezing causes cellular water to convert to ice crystals which damage the cell structure. Cryoprotectants are supposed to act by reducing the formation of Ice crystals. For biological applications, a cryoprotectant must be able to penetrate cells and have low toxicity. Compounds with such properties include glycerol, dimethyl sulfoxide, ethanediol, and propanediol. In simplest terms, cryopreservation enables cells to be stored thus avoiding the need to have all cell lines in a culture at all times. This drastically enhances the lifespan of a cell. In addition following advantages are offered.
  • Low risk of microbial contamination and cross contamination
  • Avoiding genetic drift and morphological changes 
  • Reduced costs
There are several factors that affect cell viability in cryopreservation. But the most important determining factor is the type of cell to be preserved. Some cells are particularly hard and resistant whereas certain cells are highly sensitive to changes.

The Intracellular water content is dependent on the solute concentration of cells. Anything that effects this solute concentration thus also affects the Ice crystal forming properties. It is known for a long time that the rate of change of temperature controls the transport of water across the cell membrane. The solute in the environment of the cell also contributes to this factor. Together they indirectly influence the probability of intracellular freezing and formation of ice crystals. So if we can control the cellular water levels or external osmotic balance we can avoid ice crystal formation.

Fig 1: Survival of three different cell types.
If the water permeability of the cell membrane and the temperature coefficient of water permeability is known, then it is possible to predict the effect of cooling rate on cell survival. In most cases intracellular freezing is unlikely at 1°C/min. Fig 1, depicts Survival of three different cell types frozen at various cooling rates in 1M DMSO. The figure illustrates that the cooling rate is not a universal value, rather depends on the cell type.

Table 2: CPA classification.
Cryoprotective agents (CPA) are divided into two classes- Intracellular agents and Extracellular agents. A summary of the classification and description is shown in Table 2. In general, best freezing is obtained with a combination of both types. For example in Storing PBMCs, Ficoll remnants act as Extracellular agent and DMSO is used as an Intracellular agent.

There is always some loss of cell viability after cryopreservation. Research is on to identify better CPA's. The new generation of chemicals such as Polyampholytes has shown superior properties in comparison to conventional CPA's (Glycol derivatives). Researchers have also studied Proteins found in cold regions (Commonly known as Antifreeze proteins) for their ability to preserve cells in cold temperatures.

John G. Day, & Glyn N. Stacey (2007). Cryopreservation and Freeze-Drying Protocols Humana Press. Methods in molecular biology. ISBN: 978-1-59745-362-2

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