GC-13. Comparative assessment of cryopreservation conditions for K562 and mesenchymal stromal cells cultures
Irina A. Sidorova, Kirill V. Lepik, Albert R. Muslimov, M. A.Trofimov, Olga S. Epifanovskaya, Vladislav S. Sergeev, Alexander D. Kulagin
RM Gorbacheva Research Institute, Pavlov University, St. Petersburg, Russia
Dr. Irina A. Sidorova, e-mail: firstname.lastname@example.org
Cryopreservation is a crucial step in the cell lines culturing, as well as in the production of cellular therapy products (CTP). Currently the elimination of xenogenic components of cryopreservation media is considered as a necessary condition for CTP development. The aim of this study is to compare a set of cryopreservation conditions for cell line and primary cell cultures and their effect on the viability and proliferative activity of cells.
Materials and methods
The study was carried out on the K 562 cell line and primary murine mesenchymal stromal cells (mMSC). The study included 5 groups differing in the composition of the cryopreservation medium: a) standard conditions – complete medium (CM, RPMI/®-MEM+10%FBS)+10%DMSO using a cryo container (Mr. Frosty, Nalgene, USA), b) CM+10%DMSO without use of a cryocontainer, c) commercially available xeno-free medium CryoStor® CS10 (Stem Cell Technologies, USA), d) SSP+ solution (MacoPharma, France) with 10%DMSO, e) negative control CM without DMSO. Each sample from the group was tested in three repeats, subjected to cryopreservation for 7 days at -80°C, in three independent experiments. After thawing, the cell viability was assessed by flow cytometry with the 7AAD vital staining. The dynamics of the proliferative activity of 1×10^4 cells of each of the studied groups was analyzed by the spectrophotometric method using the alamar blue vital dye after 24, 72 and 168 hours. Statistical assesment of the groups was performed with the Man-Whitney U test.
Assessment of K562 cells viability after thawing, demonstrated the median proportion of 7-AAD(+) cells in group a) of 1.5% (1.2-2.1), group b) 1.6% (1.1-1, 8) group c) 1.4% (1.2-1.9), group d) 1.4% (1.1-1.8), group e) 4.5% (2.7-5,6). Comparison of viability showed no differences between conditions a, b, c, d. The listed conditions had a statistically significant advantage over the negative control (e) (p=0.02). When assessing the viability of mMSC cells, the median proportion of 7-AAD + cells in group a) was 43.8% (16-62), b) 46.0% (22-65), c) 30.2% (17-46), d) 19.7% (16-26), e) 90.1% (86-97). The comparison revealed a statistically significant advantage of conditions a, b, c, d compared to control e (p <0.001). The number of 7AAD+ cells after conservation using SSP+ based medium was significantly lower compared to other conditions of conservation (p <0.05). Assessment of K562 cell line proliferative activity, showed an increase in the median value of the fluorescence intensity of resorufin after 168 hours compared to the value after 24 hours in group a) by 39% b) by 61% c) by 67% d) by 44% e) by 23 %. All conditions of conservation had an advantage over the negative control group (p <0.0001). The fluorescence score was significantly higher in the Cryostor group compared to standard conditions (complete medium + DMSO).
The performed analysis demonstrates no advantages of a cryo container use for cryopreservation of the studied cell types. Cryopreservation using Cryostor® CS10 medium and SSP+ based medium demonstrated the greatest potential in terms of maintaining the viability and proliferative activity of the studied cultures.
Cryopreservation, cell cultures, cell products, DMSO.