Objective: To study the research method of cell survival rate at the procedure of cryopreservation of tissue engineered tendons.
Methods: In the 4th generation of human fibroblasts, the dead cells were stained with propidium iodine (PI), while the living cells with Hoechst 33342 (Ho). The living cells and dead cells emitted fluorescence of red and blue respectively after they were stimulated by suitable ultra-violet, then flow cytometry was applied to distinguish them. The seeding cells were collected to make them to be the cell suspension of suitable concentration (6.0 x 10(5) cell/ml) before they were divided into two parts. We cryopreserved and defrosted one part three times to kill the cells and did not cryopreserve the other part, then we made cell suspension at different ratios of cryopreserved cell to non-cryopreserved cells. The fluorescence staining and flow cytometry were used to study the correlation between cell ratios of cryopreserved cell to non-cryopreserved cell and the cell survival rates. We compared the cell survival rates between immediate flow cytometry and that 2 hours after fluorescence staining.
Results: The results of flow cytometry showed that correlation between the ratio of cryopreservation and the cell survival rate was significant (r = 0.970, P < 0.05), image analysis study also showed the correlation was significant (r = 0.982, P < 0.05). The cell survival rate decreased by use of flow cytometry two hours after fluorescence staining, but there was no significant difference when compared with that of immediate flow cytometry (P > 0.05). We could also observe the cells on the tissue engineered tendons by fluorescence image directly.
Conclusion: Flow cytometry and fluorescence image after PI and Ho staining is a good way in study cell survival rate at the procedure of cryopreservation of tissue engineered tendons.