Maria Thompson

Cryopreservation and thawing of cells.

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. Curr. Protoc. Immunol. 99:A.3G.1‐A.3G.5.

Standardized Cryopreservation of Human Primary Cells

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.

Standardized Cryopreservation of Pluripotent Stem Cells

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.

Standardized Cryopreservation of Stem Cells

Successful commercial and clinical application of stem cells requires robust and practical cryopreservation protocols. Stem cells, particularly human embryonic stem cells and induced pluripotent stem cells, are notoriously sensitive to cryopreservation, requiring specialized protocols to maintain optimal cell viability and recovery. This chapter reviews the current state of stem cell cryopreservation and provides a clinically relevant, optimized method for controlled rate freezing and thawing of human stem cells that is reliable, inexpensive, and user friendly. This method successfully prepares stem cells for long-term cryogenic storage while ensuring maximal post-thaw cell viability.

Technological developments for small-scale downstream processing of cell therapies.

Despite considerable regulatory and clinical hurdles, the development and use of cell-based therapies are gaining momentum. As more of these therapies move toward commercial approval and larger-scale distribution, associated manufacturing and processing technologies are being advanced. Modern technologies directed at downstream processing seek to distribute such therapies from the manufacturing site to the patient more efficiently and reliably. Novel small-scale downstream solutions boost the transformation of cell therapies from abstraction to reality.