자가면역성 감각신경성 난청 환자에서 냉동보관법을 이용한 내이 조직 항원에 대한 T세포 반응성 검사의 유용성

Objective: Cryopreservation is very useful method for long term storage of various samples. Author tried to use the cryopreservation technique with ELISPOT assay to exam the T cell responsiveness to inner ear specific antigen (Cochlin) and evaluate its efficacy. Methods: Isolated peripheral blood mononuclear cells (PBMC) were suspended with freezing medias (A & B) in cryotube. After pre-chilling for 24 hours, transferred to liquid nitrogen tank. Cell viability and functionality were evaluated by viable cell count with trypan blue stain, proliferation assay with methyl-³H thymidine and the frequencies of IFN- γ and IL-5 producing T cells using ELISPOT assay with anti CD3 antibody before and after freezing. And also compared the frequencies of cytokine producing T cells in response to human Cochlin using ELISPOT assay with frozen cells between patient and control subjects. Results: The rate of viable cell count and stimulation index of feezed cells showed about 75%, 70% respectively when compared with before freezing cells. The frequencies of IFN- γ and IL-5 producing T cells in response to anti CD3 were 68% and 71% of before freezing condition. And the frequencies of IFN- γ and IL-5 producing T cells response to human recombinant cochlin were significantly higher in patients than control subjects using cryopreserved cell. Conclusion: The functional responsiveness and cell number of cryopreserved cells were restored nearly before freezing level. So the cryopreservation technique could be very useful tool in ELISPOT assay for determination of the frequencies of cytokines. (J Clinical Otolaryngol 2007;18:170–177)