NutriFreez™ D10 Cryopreservation Medium - Performance Validation

When using serum-free media in cell culture, it is important to cryopreserve cells also in a medium free of serum. The novel cell freezing medium that has been developed by Biological Industries contains no serum, but rather methylcellulose and DMSO. NutriFreez™ D10 Cryopreservation Medium is complete, ready to use solution which is designed to protect frozen cells in liquid nitrogen for long-term storage.

Materials and methods

Cell Lines

Various cells grown under serum-free conditions were frozen with serum-free freezing medium and with freezing medium containing serum

Freezing Method

NutriFreez™ D10 Cryopreservation Medium (Cat. no.: 05-713-1) containing 10% DMSO and Methylcellulose, and basal medium containing 10% DMSO and 20% FBS were used as freezing media. The cells were frozen in the appropriate freezing medium in a concentration of 3-5x106 per ml. One ml of these cell suspensions was transferred to a plastic ampoule and frozen by decreasing the temperature at a rate of 1-2oC/min. The ampoules were kept in liquid nitrogen until tested.

Cell recovery measurements

When thawing, the frozen ampoules were put in a water bath at 37°C. After dilution with culture medium and centrifugation, the cells were resuspended with either serum-free medium or medium containing serum. Viability of cells was determined by the trypan blue dye exclusion method. Adhesion of cells was determined by counting the attached cells only 6-24 hours after culture of the cells.

Results

Freezing of cells in NutriFreez™ D10 Cryopreservation Medium in comparison to freezing medium containing serum.

Table 1: Comparison of NutriFreez™ D10 Cryopreservation Medium to freezing medium containing serum.

Cell Line% Viability  % Adhesion
 NutriFreez™ D10 Cryopreservation MediumFreezing Medium Containing SerumNutriFreez™ D10 Cryopreservation MediumFreezing Medium Containing Serum
3T3 85 83 100 83
BGM 91 83 88 88
VERO 66 71 62 33
HEp-2 75 69 100 92
BSC-1 82 77 22 100

Long term storage of cells in liquid nitrogen using NutriFreez™ D10 Cryopreservation Medium.

Table 2: Recovery of cells frozen in NutriFreez™ D10 Cryopreservation Medium

 24 Hours Storage 6 Months Storage 4 Years Storage
Cell LIne% Viability% Adhesion% Viability% Adhesion% Viability% Adhesion
B16-F10 85 100 74 79 72 80
BGM 80 70 61 100 66 92
BHK-21 80 93 64 100 71 95
CHO-DHFR 70 70 69 81 70 73
HELA 97 78 70 90 80 83
HEp-2 90 100 63 100 66 94
MA-10 88 95 81 100 83 91
VERO 90 94 71 68 73 76

Summary

NutriFreez™ D10 Cryopreservation Medium supports efficient cryopreservation of various cell lines cultured in serum-free media.

After freezing and thawing, a very high percentage of viable cells is obtained, and they also show excellent attachment ability as well as growth performance. In fact, the present study has shown that in most cases higher viability and adhesion percentages are obtained in comparison to freezing medium containing serum. Therefore, the use of NutriFreez™ D10 Cryopreservation Medium is also recommended for cell culture employing serum-supplemented growth media.