THE EFFECTS OF A SIMPLE METHOD FOR CRYOPRESERVATION AND THAWING PROCEDURES ON CORD BLOOD DERIVED DC-BASED ESOPHAGEAL CARCINOMA VACCINE RUNNING TITLE: CRYOPRESERVATION OF FUSED CARCINOMA VACCINE

BACKGROUND: Producing sufficient numbers of DCs at one time point and subsequently cryopreserving the generated DCs in ready-for-use aliquots for clinical application is useful in cancer treatment. OBJECTIVE: To study the effects of a simplified cryopreservation method and thawing procedures acting on the biological characteristics and specific cytotoxic activity of cord blood derived DC-based esophageal carcinoma vaccine.. MATERIALS AND METHODS: CD34+ hematopoietic stem cells were isolated from cord blood using CD34+ Progenitor Cell Isolation Kit by magnetic cell sorting system (MACS). The CD34+ cells were expanded with cytokines as DCs, and fused with EC109 cells by PEG 3600. The fused cells were transferred to a freezing tube without rate-controlled freezing and stored at -80 degrees C for three weeks. During cryopreservation, 2.5% DMSO, 2.5% glucose and 10% FCS at final concentration was used as stock solution. After thawing, cells were assayed for Typan blue viability, morphology, immunophenotypes and T-cell stimulatory capacity, and specific CTL activity. RESULTS: Cryopreservation does not cause significant changes in the phenotypes expression or morphology of the fused cells, and the viability were well preserved (Typan blue viability was 77.2 +/- 1.8%). After being stimulated by DC-based esophageal carcinoma vaccine either before or after cryopreservation, the numbers of CD3+T/CD4+T and CD3+T/CD8+T lymphocytes increased obviously, especially for CD3+T/CD4+T, and the ratio of CD4/CD8 changed from 0.85 to 1.29 and 1.25 respectively. Specific CTL activity were well preserved (compare to the fresh fused vaccine, P>0.05). CONCLUSION: A simple -80 degrees C freezing and storage method is practical for cord blood derived DC-based esophageal carcinoma vaccine. It will greatly facilitate the clinical use of DC-based vaccine for immunotherapy.