Cryopreservation of equine mononuclear cells for immunological studies.

This research paper presents a quick and efficient method for storing and subsequently recovering equine mononuclear cells. Even after cryopreservation, these cells were found to exhibit nearly the same functional attributes as those directly derived from fresh samples.

Methods of Cell Preservation and Recovery

• The researchers developed a method to freeze buffy-coat leukocytes, which are a mix of white blood cells and platelets, in the horse’s own plasma tempered with 10% DMSO (Dimethyl sulfoxide). DMSO is commonly used in cell cryopreservation to prevent crystallization — which could kill or harm the cells during the freezing process.
• Upon thawing, the mononuclear cells were isolated from the whole leukocyte population by a gradient sedimentation process using a routinely employed Ficoll-Hypaque purification process. This method makes use of the different densities of blood cells to separate the various types.

Recovery Efficiency and Functionality of Cryopreserved Cells

• The recovery rate of mononuclear cells (which include types such as lymphocytes and monocytes) from the cryopreserved samples was impressively high, between 82% – 94% of those obtained from fresh blood samples. Thus, a considerable majority of the cells survived the freeze-thaw process effectively.
• The functionality of these cryopreserved and recovered cells was evaluated by assessing their capability to proliferate, or multiply, when exposed to certain stimulants known as mitogens and specific antigens. The response of the cryopreserved cells matched those of the fresh ones. They reached the same maximum level of proliferation, though, interestingly, the cryopreserved cells usually took an extra day in culture to reach those peak proliferation levels.

Evaluation of Surface Antigen Expression

• The expression of cell-surface antigens was evaluated using Flow cytometry technique, which allows the analysis of multiple physical characteristics of a single cell. Surface antigens are proteins expressed on the outer surface of the cells and are critical for immune response as they can be recognized by immune cells. The study found no significant effect of the cryopreservation process on the antigen expression of the cells.

Overall Utility and Advantages of the Technique

• The process described in this study offers a simple, rapid, and efficient method for the preservation of large numbers of leukocytes that can be assessed for various immune functions at a later time. This ability to preserve cells without loss of functionality expands the possibilities of immunological studies, offering a broader timeframe for analysis. This is especially helpful when the availability of fresh samples might be restrictive.