Hu Yong, Li Lei, Yu Yin, Huang Haishui, Uygun Basak E., Yarmush Martin L.
Regenerative therapies require availability of an abundant healthy cell source which can be achieved by e±cient cryopreservation techniques. Here, we established a novel approach for improved cell cryopreservation using an mRNA-based dCas9-VP64 gene activation system for transient, yet highly e±cient expression of epigenetic related genes in mammalian cells for repression of metabolic activity. Before freezing, mammalian cells were treated by dCas9-VP64- modi¯ed mRNA and guide RNAs for upregulation of histone deacetylase (HDAC), DNA methyltransferase (DNMT) and transcriptional co-repressor Sin3A genes. Cell viability, karyotype, pluripotency, and other cell speci¯c functions were analyzed during post-thaw culture. Using conventional cryopreservation protocols, we found improvement of viability in dCas9- VP64 pretreated cells (P < 0:05) compared to untreated cells. Combined with dCas9-VP64 system, a reduced amount of cryoprotectant (5% DMSO) did not negatively a®ect the post-thaw viability. Co-delivering chemically modi¯ed dCas9-VP64 mRNA with gRNAs is an e±cient gene delivery method compared to DNA-based strategies, without the associated safety concerns. This approach is a simple, yet e®ective way to accelerate a wide array of cellular research and translational medical applications.