Surface‐based cryopreservation strategies for human embryonic stem cells: A comparative study

Abstract

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. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1079–1087, 2012

Toxicity Minimized Cryoprotectant Addition and Removal Procedures for Adherent Endothelial Cells

“…The ability to cryopreserve adherent cells would also allow off-the-shelf availability for applications such as drug screening [ 4 ] and cell-based biosensors [ 5 ]. Conventional slow cooling approaches have been used previously to cryopreserve adherent cells [ 1 , 2 , 6 – 10 ], but cell recovery post-thaw has typically been low, and it has been suggested that cell-cell and cell-substrate connections make adherent cells particularly susceptible to freezing damage [ 11 – 13 ].…”

“…Ice-free cryopreservation, known as vitrification, is a cryopreservation procedure that prevents ice crystal formation in the entire system, not just in the intracellular space, and is a promising method for cryopreservation of adherent cells and tissues [ 6 , 14 , 15 ]. Ice-free cryopreservation requires a balance of extremely high cooling and warming rates and high CPA concentrations.…”

Directional freezing for the cryopreservation of adherent mammalian cells on a substrate

“…We suggest that the cooling rate in cell culture cryopreservation should be slowed down with the decrease of temperature, which is also in line with a protocol applied in embryo cryopreservation [ 36 ]. Our approach displays high reproducibility and high survival rates compared to non-directional slow freezing methods and other previously reported attempts of cryopreservation of cells in an adherent state [ 3 , 12 – 18 , 21 ]. Our method can be readily applied as a robust cryopreservation protocol for various in vitro applications.…”

Drug-resistant colon cancer cells produce high carcinoembryonic antigen and might not be cancer-initiating cells

“…Conventional staining protocol35–39 was used. The expression of CD133, forward scattering intensity, and side scattering intensity of the LoVo cells were analyzed by flow cytometry (Coulter EPICS™ XL; Beckman-Coulter, Brea, CA, USA).…”