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 1-10x106 cells/ml per vial. 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 Medium | Freezing Medium Containing Serum | NutriFreez® D10 Cryopreservation Medium | Freezing 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.
