"Cryopreservation" is the most common process in the biological field. In cell culture procedures, it is sometimes necessary to store the cells to be used for subsequent research or use in cell therapy, which can be saved more cells for the patient. In the freezing process, the unstable changes caused by freezing have different effects on the cells. If the operation is careless, it is very likely that the cells will be inadvertently necrotic and fall short during freezing. Therefore, scientists attach great importance to this and must study samples for different cell lines with different solution formulations to create the best results.

Continuing the previous phase of cell therapy, this phase will introduce the relevant precautions when cells are cryopreserved: Since the preservation of cells in vitro requires extremely high stability and strict environmental requirements to survive, in order to preserve the cells for a long time, freezing technology is used to store the cells in liquid nitrogen, so that the cell metabolic reaction enters the resting phase. Defrost it again at a time. In the process of freezing and thawing, the culture medium will encounter problems such as crystallization, solute precipitation, changes in polarity, changes in pH, and changes in concentration. Here we will provide solutions for crystallization, concentration changes, and pH changes.

Further Reading: The application of Cell Therapy

Crystallization:

Slow freezing:

During the freezing of cells, if the water molecules in the cells are too late to flow out, they will enter the glass transition state, which will cause the water molecules in the cells to form large ice crystals, which will cause the cells to rupture and die. To solve this problem, there are currently two ways: Slow freezing method: through slow freezing, It had enough time for the water molecules in the cells to be discharged, and cryopreservatives are added to prevent the water molecules inside and outside the cells from forming too large ice crystals so that they can be completely dehydrated as much as possible.

Vitrification freezing:

Add a higher concentration of cryopreservative to the medium, and use the rapid freezing method to quickly replace the water in the cells in a short time, so that the liquid in the cell becomes highly viscous, high-concentration vitrified substance to reduce ice crystals The damage caused. However, high-concentration cryopreservatives may be toxic to cells, so the operation must be fast.

In the literature we found, it was proposed to use the Hammett acidity function (Hx) to measure the acidity of a more concentrated solution (Hammett and Deyrup 1932). Since H2− depends not only on the proton activity but also on the ratio of the activity coefficient of the indicator (Govindarajan et al., 2006; Pudipeddi et al., 2008). Therefore, in principle, the Hx values can be different from each other in various indexes. However, since the applied indicators belong to the same sulfophthalein family, based on previous observations (Yi Yao 1998, Vetr´akova´ et al., 2017), we expect internal consistency between the indicators. Therefore, we will introduce the acid value in this article with H2−

HEPES:

The result of mixing Na-P (20 – 100 mM) with 50 mM HEPES solution; no major acidity changes were observed in the expected concentration range. In the literature, two Na-P concentrations of 5mM and 50 mM are used to compare different HEPES concentrations (0~30 mmol L-1) (fig 3), compared with the sample without Na-P (fig 2.) The change of H2⁻ value is very obvious.

MOPS:

In the mixed result of 20 mM MOPS and 50 mM Na-P, the H2- value increased to (7.49 ± 0.0503), and the acidity change was relatively stable.

MES:

In order to minimize pH changes, the required MES concentration is approximately 12 mM in 5 mM Na-P and approximately 80 mM in 50 mM Na-P.

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Reference:

Making good’s buffers good for freezing: The acidity changes and their elimination via mixing with sodium phosphate / Luk´aˇs Veselý, Behera Susrisweta, Dominik Heger

Comparison of vitrification and conventional cryopreservation of day 5 and day 6 blastocysts during clinical application Juergen Liebermann 1 , Michael J Tucker Affiliations / PMID: 16762345 DOI: 10.1016/j.fertnstert.2006.01.029