Successful cryopreservation of cells requires not only that the cells be handled in a proper fashion for harvesting with equipment in place to ensure consistency, reproducibility, and sterility, but also that a correct choice and amount of cryoprotective agent is added. In general, a controlled freezing rate of 1°C/min is necessary to retain optimal viability of the recovered cells. There are many variations of cell freezing methods in use, including costly electronically regulated control rate freezers, unstandardized, passive isopropyl alcohol freezing containers, and crude rudimentary devices constructed from Styrofoam boxes or paper insulation. However, for the freezing and recovery of cell lines, primary cells, and stem cell cultures, the protocol described in this unit is simple, reproducible, and successful. Not only does it eliminate the need for isopropanol, as well as the costs and hazards associated with its use and disposal, but it provides a uniform method with improved cell viability and recovery.
Cryopreservation is the use of low temperatures to preserve structurally intact living cells. The cells that survive the thermodynamic journey from the 37°C incubator to the −196°C liquid nitrogen storage tank are free from the influences of time. Thus, cryopreservation is a critical component of cell culture and cell manufacturing protocols. Successful cryopreservation of human cells requires that the cells be derived from patient samples that are collected in a standardized manner, and carefully handled from blood draw through cell isolation. Furthermore, proper equipment must be in place to ensure consistency, reproducibility, and sterility. In addition, the correct choice and amount of cryoprotectant agent must be added at the correct temperature, and a controlled rate of freezing (most commonly 1°C/min) must be applied prior to a standardized method of cryogenic storage. This appendix describes how human primary cells can be frozen for long‐term storage and thawed for growth in a tissue culture vessel. Curr. Protoc. Cell Biol. 64:A.3I.1‐A.3I.8. © 2014 by John Wiley & Sons, Inc.
Cryopreservation is the process by which intact living cells are preserved at cryogenic temperatures in liquid nitrogen. Freezing cells allow them to be stored, often for years, in a state where their normal metabolic activity is suspended in order to protect them from damage due to chemical reactivity and time. The cells can then be thawed and resuscitated as needed. Cells are cryopreserved in order to guard against genetic drift in continuous cell lines, or against transformation or differentiation in noncontinuous cell lines, such as stem cells and primary cells. In the case of primary cells that are isolated directly from the tissue of interest, they have a finite ability to divide; therefore, cryopreservation is necessary to preserve their unique characteristics for future experiments. Optimal cryopreservation of cells relies on proper freezing and thawing methods. To be protected from structural damage during the freeze‐thaw process, mammalian cells are frozen in the presence of cryoprotectant. Post‐thaw viability assays are then conducted to measure the success of the cryopreservation techniques by calculating the percentage of frozen cells that are alive and able to recover normal function once thawed. Key Concepts: Cryopreservation is used to enable long‐term storage, preserve current genetic state, prevent transformation or differentiation during subculture and provide a back‐up stock in case of infection/contamination during culture. A controlled rate of freezing and rapid thawing is necessary for optimal cryopreservation and recovery. Care must be taken to minimize transient warming events during transfer and storage, as it impacts viability and recovery. Active thawing results in higher cell viability and recovery than passive thawing. Timing is critical to all stages of the cryopreservation process. All materials should be ready before beginning the procedure, and steps should be taken to ensure each sample is handled with minimal delay. Method standardisation and quality control are necessary to maintain sample integrity and reproducibility.
Cryopreservation is the use of very low temperatures to preserve structurally intact living cells. Mammalian cells are cryopreserved to avoid loss by contamination, to minimize genetic change in continuous cell lines, and to avoid transformation in finite cell lines. The cells which survive the
The successful exploitation of human cells for research, translational, therapeutic, and commercial purposes requires that effective and simple cryopreservation methods be applied for storage in local and master cell banks. Of all the cell types utilized in modern research, human embryonic stem cells and their more recent relatives, induced pluripotent stem cells, are two of the most sensitive to cryopreservation. It is frequently observed that the lack of quality control and proper processing techniques yield poor recovery of pluripotent stem cells. The procedures in this unit have been optimized for handling some of the most recalcitrant stem cell lines, and provide a method for controlled‐rate freezing, using minimal equipment that affords levels of cell viability comparable to expensive controlled‐rate freezers. The protocol also eliminates the requirement for isopropanol, avoiding the hazards, on‐going cost, and inconsistencies associated with its use and disposal. It provides a clinically relevant, inexpensive, reliable, and user‐friendly method that successfully prepares cells for long‐term cold storage and ensures maximum levels of cell viability post thaw. Curr. Protoc. Stem Cell Biol. 28:1C.14.1‐1C.14.10. © 2014 by John Wiley & Sons, Inc.
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