Question

Describe 3 sources of human error associated with the addition of CPA’s and the freezing process and explain how the errors will affect the cells

Answer 1

Answer:

Understanding the role of water and the need to adequately remove it from cells or abrogate its ability to form ice crystals, which damage the cell membrane, is critical to successful cryopreservation. When cells are frozen in aqueous suspension, often they are destroyed.

However, in the 1940s Polge and others discovered the cryoprotective properties of glycerol. Since then several chemicals, generically called cryoprotectant agents (CPAs), have been identied. The mechanism of action of CPAs is complex and is not fully understood.

However, according to the commonly accepted theory of colligative action, CPAs increase solute concentration both within the cell and extracellularly, thereby suppressing ice formation. For this purpose, the so-called penetrating (or intracellular) CPAs [e.g., dimethylsulfoxide (DMSO), glycerol, propanediol, and methanol] must be able to cross the cell membrane readily and penetrate the cell without signicant toxicity.

There also is a group of nonpenetrating (or extracellular) CPAs (e.g., sucrose and trehalose) whose mechanism of action is thought to be related at least in part to their stabilizing interaction with cell membranes. This property also may explain the cryoprotective activities of certain large molecular weight compounds such as hydroxyethyl starch and polyvinylpropylene. Theoretical models of cryoprotection typically evoke the colligative theory, but full explanation of CPA action is yet to be established.

An alternative form of cell preservation, commonly called vitrication, whereby the cell suspension is loaded with high levels of penetrating CPAs (often several in combination), induces a glass-like state in which cellular and extracellular water cannot readily form ice crystals. When cell suspensions prepared in this way then are cooled very rapidly (cooling rates of 100°–1000°/min or more) the extreme viscosity prevents osmosis, and the water molecules are unable to form ice.

This procedure has been widely used for complex structures including a variety of human, plant, and animal tissues and may help preserve those cell preparations that have variable degrees of cellular permeability or when standard cryoprotection cannot deliver the range of conditions required to optimally preserve viability in all the tissues' component cell types.

CPAs have biological activities beyond their cryoprotective properties. Some, like DMSO, can affect the cell membrane, cytoskeleton, and gene expression and may be toxic to cells following prolonged exposure. Therefore, during development of new cryopreservation protocols analysts should perform a toxicity assay in which the cells are exposed to the CPA over a range of time intervals to evaluate loss of viability or alteration of functionality.