Beschreibung
Cryopreservation is a process that allows cells, organelles, and tissues to be preserved over a longer period of time by cooling to exceptionally low temperatures. The aim of this study was to investigate the influence of cryopreservation and various freezing media on vitality, differentiation and structural preservation of precision incisions (PCLS) of lung tissue. The temporal limitation of PCLS requires a possibility to store them over a longer period with-out affecting functionality. The usability of PCLS after cryopreservation was verified by various vitality tests. For cryopreservation, freezing media were tested, which received the cryoprotective agents DMSO and trehalose at different concentrations. After establishing the optimal freezing method on murine PCLS, this was transferred to human tissue. Vitality was determined by LDH release, MTT-Assay, and live/dead fluorescence staining. The results showed that the freezing medium exerts an influence on vitality, the best vitality could be achieved with DMEM/F-12 + 1 % penicillin/streptomycin + 10 % FKS + 10 % DMSO + 200 mmol/l trehalose.
The comparison between fresh and cryopreserved PCLS showed that both, viability and metabolic activity, were reduced after cryopreservation. The structural integrity of the PCLS also demonstrated a negative change after cryopreservation and at the protein level no in-creased expression of the cleaved caspase-3 was observed, which was used as a marker of apoptosis induction. In a further step, the surfactant production of PCLS as a function of the media and cryofixation was investigated. For this purpose, fluorescence staining with LysoTracker Green and a measurement of the surfactant producing proteins B and C were performed using Western blot methodics. It was found that cryopreserved PCLS still have differentiated alveolar epithelial cells type II and to some extent SP-B and SP-C are still detectable, but there was a reduction compared to unfrozen PCLS.
The results show that cryopreservation and long-term storage of PCLS are principally a promising solution to the limited viability of PCLS in culture. It has been demonstrated that both murine and human lung tissue can be vitally cryopreserved. However, it seems useful to carry out further studies to improve the viability after cryopreservation and to protect the PCLS even more effectively from damage.
