125 - Off the Shelf Cellular Therapeutics: Factors to Consider During Cryopreservation and Storage of Human Cells for Clinical Use

Abstract

The field of cellular therapeutics has immense potential, affording an exciting array of applications in unmet medical needs. One of several key issues is an emphasis on getting these therapies from bench to bedside without compromising safety and efficacy. The successful commercialization of cellular therapeutics will require many to extend the shelf-life of these therapies beyond shipping “fresh” at ambient or chilled temperatures for “just in time” infusion. Cryopreservation is an attractive option and offers potential advantages such as storing and retaining patient samples in case of a relapse, banking large quantities of allogeneic cells for broader distribution and use, and retaining testing samples for leukocyte antigen typing and matching. However, cryopreservation is only useful if cells can be reanimated to physiological life with negligible loss of viability and functionality. Also critical is the logistics of storing, processing and transporting cells in clinically appropriate packaging systems and storage devices consistent with Quality and Regulatory standards. Rationalized approaches to develop commercial-scale cell therapies require an efficient cryopreservation system that provides the ability to inventory standardized products with maximized shelf life for later on-demand distribution and use, as well as a method that is scientifically sound and optimized for the cell of interest. The objective of this paper is to bridge this gap between the basic science of cryobiology and its application in this context by identifying several key aspects of cryopreservation science in a format that may be easily integrated into mainstream cell therapy manufacture. Some of the key damage mechanisms and associated factors to consider described above are summarized in Table I. It is important to also consider that in developing a cryopreservation process, variables should be adjusted not just based on total cell recovery and gross viability, but on functional recovery, with the specific product efficacy requirements in mind to define what “functional” means to that specific cellular therapeutic product. If a robust cryopreservation and post-thaw processing system is developed, the product shelf life is essentially indefinite so long as temperature is maintained. Table ISummary of definitions of damage mechanisms and factors to consider associated with a cryopreservation process. Damage Mechanism Description and Factors to Consider Osmotic Injury or Toxic injury Injury due to the addition and removal of cryoprotective agents; cell specific characteristics such as biophysical parameters (size, shape, membrane permeability to water and cryoprotectants, osmotically inactive water, osmotic and volumetric tolerance limits) should be considered. Cold-Shock Injury Injury due to an abrupt change in temperature; cooling rate should be considered, with very slow (<0.5°C/min) cooling rates applied to cold-shock sensitive cells. Chilling Injury Injury due to prolonged exposure to cold (but above cryogenic) temperatures; absolute exposure time is the most critical factor to consider. If cells appear to be chilling sensitive but are tolerant of a cryoprotectant such as DMSO at warmer temperatures, strategies can be employed to perform cryoprotectant additions at or near room temperature and reduce the amount of time “chilled”. Cooling Injury Injury associated with extracellular and intracellular ice formation; factors to consider can be cell-type specific and include cooling rate, ice nucleation regimen, supercooling, end temperature prior to transfer to storage, cellular dehydration, intracellular ice formation, and hypertonic solute toxicity. Storage Injury Injury due to unwanted thermal fluctuations (transient warming events), cosmic rays and free radical formation; factors to consider include the glass transition temperature of the cryoptrotectant and careful maintenance of the storage temperature at all times. Properly cryopreserved and stored cells are viable indefinitely. While practically challenging, if at all possible a sample should never be removed from cryostorage until it is to be used, otherwise temperature of the sample should be monitored throughout any temporary removal (such as removing a rack of vials or frame of bags). Additional considerations should include the use of closed system containers for storage (in vapor or liquid). Thawing injury Injury associated with warming sample from LN2 storage temperature to above phase change temperature; potential recrystallization during warming should be considered. If slow cooling is used, a wide range of warming rates are likely acceptable, however in general faster warming may result in less intracellular recrystallization. Post-cryopreservation Processing Upon thaw, cells are in a potentially compromised state, care must be given to appropriately prepare them for use. If a permeable cryoprotectant is used (such as DMSO), knowledge of cell-specific osmotic characteristics is important. Cells swell and may lyse upon removal of permeable cryoprotectants and may not survive 1-step dilution. If cells are administered directly from thaw without dilution or a washing step, this is effectively a 1-step dilution and may result in significant cell loss in vivo. Open table in a new tab