Simple, fast and efficient iTOP-mediated delivery of CRISPR/Cas9 RNP in difficult-to-transduce human cells including primary T cells.

The advent of the CRISPR/Cas9 system has transformed the field of human genome engineering and has created new perspectives in the development of innovative cell therapies. However, the absence of a simple, fast and efficient delivery method of CRISPR/Cas9 into primary human cells has been limiting the progress of CRISPR/Cas9-based therapies. Here, we describe an optimized protocol for iTOP-mediated delivery of CRISPR/Cas9 in various human cells, including primary T cells, induced pluripotent stem cells (hiPSCs), Jurkat, ARPE-19 and HEK293 cells. We compare iTOP to other CRISPR/Cas9 delivery methods, such as electroporation and lipofection, and evaluate the corresponding gene-editing efficiencies and post-treatment cell viabilities. We demonstrate that the gene editing achieved by iTOP-mediated delivery of CRISPR/Cas9 is 40-95 % depending on the cell type, while post-iTOP cell viability remains high in the range of 70-95 %. Collectively, we present an optimized workflow for a simple, high-throughput and effective iTOP-mediated delivery of CRISPR/Cas9 to engineer difficult-to-transduce human cells. We believe that the iTOP technology® could contribute to the development of novel CRISPR/Cas9-based cell therapies.

Efficient cryopreservation of human pluripotent stem cells by surface-based vitrification.

Efficient cryopreservation of human stem cells is crucial for guaranteeing a permanent supply of high-quality cell material for drug discovery or regenerative medicine. Conventionally used protocols usually employing slow freezing rates, however, result in low recovery rates for human pluripotent stem cells due to their complex colony structure. In this chapter, a surface-based vitrification protocol for pluripotent stem cells is presented based on a procedure for human embryonic stem cells developed by Beier et al. (Cryobiology 63:175-185, 2011). This simple and highly efficient cryopreservation method allows cryopreservation of large numbers of ready-to-use adherent cells that maintain pluripotency.

Surface-based cryopreservation strategies for human embryonic stem cells: a comparative study.

Human embryonic stem cells (hESC) hold tremendous potential in the emerging fields of gene and cell therapy as well as in basic scientific research. One of the major challenges regarding their application is the development of efficient cryopreservation protocols for hESC since current methods present poor recovery rates and/or technical difficulties which impair the development of effective processes that can handle bulk quantities of pluripotent cells. The main focus of this work was to compare different strategies for the cryopreservation of adherent hESC colonies. Slow-rate freezing protocols using intact hESC colonies was evaluated and compared with a surface-based vitrification approach. Entrapment within ultra-high viscous alginate was investigated as the main strategy to avoid the commonly observed loss of viability and colony fragmentation during slow-rate freezing. Our results indicate that entrapment beneath a layer of ultra-high viscous alginate does not provide further protection to hESC cryopreserved through slow-rate freezing, irrespectively of the cryomedium used. Vitrification of adherent hESC colonies on culture dishes yielded significantly higher recovery rates when compared to the slow-rate freezing approaches investigated. The pluripotency of hESC was not changed after a vitrification/thawing cycle and during further propagation in culture. In conclusion, from the cryopreservation methods investigated in this study, surface-based vitrification of hESC has proven to be the most efficient for the cryopreservation of intact hESC colonies, reducing the time required to amplify frozen stocks thus supporting the widespread use of these cells in research and clinical applications.