Investigation of epigenetic changes caused by cryopreservative procedures

摘要

Cryopreservation techniques allow the long term storage of a wide variety of biological materials without significant deterioration in quality. Immediate post-thaw survival is most often used to assess the effect of freeze-thaw process on cells. This parameter however provides no information on the possible subtle effects of cryopreservation, including DNA damage, alteration of mRNA levels and protein function. Exposure to dimethyl sulfoxide (DMSO) which is the most commonly used cryoprotective agent (CPA) has recently been shown to have an impact on the epigenetic profile in mouse embryoid body. Epigenetics describes heritable alterations in gene expression or cellular phenotype, occurring without changes in the DNA sequence. Epigenetic modifications affect gene expression by altering eukaryotic transcriptional regulation. Factors involved in such modifications include DNA methylation, histone modifications, RNA interference, and genomic imprinting. Mesenchymal stem cells (MSCs) from the common marmoset monkey, Callithrix jacchus were frozen in a controlled rate freezer using different concentrations of DMSO. The effect of DMSO on cell viability, proliferation, DNA methylation patterns and histone modification patterns of the different cell samples were studied. Cell viability was tested by analyzing the membrane integrity (by Trypan Blue staining) and proliferation (using MTT assay) after cryopreservation. The functional integrity of cryopreserved cells was tested by analyzing the ability of these cells to differentiate into adipocytes and osteocytes. The global DNA methylation levels of cryopreserved cells were studied using ELISA. The global acetylation and methylation patterns of the different histone proteins were analyzed using immunocytochemistry and mass-spectrometry. The results indicated that the survival and epigenetic properties of MSCs might be influenced depending on the concentration of DMSO used for cryopreservation. These results will be replicated and validated with further experimentation. Further experimentation will be performed to study the mRNA and protein expression of epigenetic proteins of marmoset MSCs exposed to pre-established optimal and suboptimal cryopreservation protocols, using PCR and western blotting.