Technical Considerations in the Freezing, Low-Temperature Storage and Thawing of Stem Cells for Cellular Therapies

Abstract

The commercial and clinical development of cellular therapy products will invariably require cryopreservation and frozen storage of cellular starting materials, intermediates and/or final product. Optimising cryopreservation is as important as optimisation of the cell culture process in obtaining maximum yield and a consistent end-product. Suboptimal cryopreservation can lead not only to batch-to-batch variation, lowered cellular functionality and reduced cell yield, but also to the potential selection of subpopulations with genetic or epigenetic characteristics divergent from the original cell line. Regulatory requirements also impact on cryopreservation as these will require a robust and reproducible approach to the freezing, storage and thawing of the product. This requires attention to all aspects of the application of low temperatures: from the choice of freezing container and cryoprotectant, the cooling rate employed and its mode of de-livery, the correct handling of the frozen material during storage and transportation, to the eventual thawing of the product by the end-user. Each of these influences all of the others to a greater or lesser extent and none should be ignored. This paper seeks to provide practical insights and alternative solutions to the technical challenges faced during cryopreservation of cells for use in cellular therapies.

Keywords: Cell therapy; Cryopreservation; Cryoprotectants; Freezing; Low-temperature storage; Stem cells; Thawing.

Fig. 1

Typical cooling curves for PCDs. PCDs were operated according to the manufacturer's instructions. Thin wire thermocouples (TWTc) were inserted through adapted PCD lids into the centre of spe­cially adapted cryovials containing 1 mL of CPA (10% DMSO in FBS). These were then placed in the PCD. The remaining spaces were filled with cryovials containing 1 mL of CPA. All cryovials were equilibrated at 20°C for between 20 and 30 min before transfer of the PCD to a monitored, controlled-access −80°C freezer. The temperature was recorded every 6 s for 5 h using a multi-channel datalogger. The illustrations show position of the TWTc. Mean cooling rate (MCR) between −10 and −40°C was based on the number of technical replicates per experiment shown on the graph. A minimum of 3 experiments were performed in each group. A PCDs Mr Frosty and CoolCell 12. B CoolCell FTS30 (note reduced cooling rate compared to the CoolCell 12). C Stacked PCDs. Two Mr Frostys were treated as above but stacked on top of each other and placed within the same compartment of the −80°C freezer. Of the 4 TWTc shown, two were housed in the upper and two in the lower PCD. Note the significantly reduced cooling rate for cryovials in the upper PCD.

Fig. 2

Effect on the temperature of cryovials, located in different positions within a cryovial box, of removing the inventory tower, containing the cryobox, from LN₂ storage. Thin wire thermocouples (TWTc) were inserted into adapted cryovials containing 1 mL of CPA and placed either at the periphery or centre of a 5 × 5 cryovial box. The boxes were either filled to capacity or remained empty. Temperature was recorded every 60 s. The cryovials rewarmed at rates between 6.8°C/min and 11.5°C/min depending on their location in the box and the degree of insulation afforded by surrounding cryovials. The illustrations show the position of TWTc.