August 2, 2023
Cryopreservation of cells is a necessary step in drug manufacturing and development which relies on a constant cell supply and cell reproducibility. By freezing cell lines at cryogenic temperatures, different cell types can be stored for future use without the risk of contamination or genetic alterations.
In the following, we will take a closer look at the process of cryopreservation, which role it plays for cell banking and which challenges have to be overcome to reach the successful cryopreservation of cells.
The definition of cryopreservation entails all processes in which biological materials like cell lines, stem cells, tissue or organs are cooled down to low temperatures to store them for a longer period of time, e.g. in liquid nitrogen. While cryopreservation means the storage of material in cryogenic temperatures, the freezing process is referred to as cryogenic freezing and brings products down to temperatures far below 0 °C.
Cryogenic freezing stops the living cell’s metabolism, ensuring cell viability during long-term storage. While the cryogenic temperature range lies between -150 °C and -273 °C, cell degradation can already be stopped at -80 °C. During freezing, cryoprotective agents (CPAs) are added as cryopreservation media to the bags to prevent potential cell death.1
Cryopreservation and cryobiology have many advantages for different applications in the pharma industry, ranging from the preservation of pluripotent stem cells for in vitro fertilization or hematopoietic tissue preserved for transplantation. The cryopreservation of cell lines has become an important part in the development of new biologics, such as vaccines, and plays a vital part in biotechnology, clinical assays and research, but also in several targeted therapies, including autologous stem cell therapy, where cell freezing is performed to preserve stem cells, e.g. harvested in the bone marrow.
Through cryopreservation, cell loss through microbial contamination can be drastically reduced. By freezing cells, it becomes possible to preserve specific cell banks as back up cells without the risk of genetic changes in the original cell lines. The purpose of cell banking lies in the reproducibility of the original frozen cells. Their clones can be used in drug compounds. As these formulations have to guarantee continuity and low toxicity, it is important to reduce these risks through cryopreservation.
Whether a cell culture is adherent or in suspension has an impact on the process that is needed to perform cryopreservation with high post-thaw viability.
Cultures in suspensions have to be counted and then added to a centrifuge, where the supernatant is removed. Afterwards, the freezing medium is added to the serum-containing medium, before aliquoting it into cryogenic storage bags. The cells have to be frozen in slow cooling rates at 1 °C per minute until they reach the desired temperature and are stored in cryogenic freezers, often based on liquid nitrogen.2
The cryopreservation protocol for freezing adherent cell cultures differs in the beginning: The cells have to be recovered from the substrate by thawing with dissociation agents first, so they can be detached without causing any damage. After they have successfully been removed, they can be added to a growth medium before they are mixed with supernatant in a centrifuge.2
Successful cryopreservation can only be executed with professional, up-to-date equipment that helps staff comply with current good manufacturing practices (cGMPs) as well as regulations set by the Food and Drug Association (FDA). Tools in cryopreservation of cells include freezing containers, labels and markers, controlled rate freezers and equipment for the thawing process like water baths. Further, a centrifuge, supernatant and cryoprotectant serum is needed for the process.
Cryoprotectant agents (CPAs) are needed to reduce solutes and thereby ice formation during cryogenic freezing, which can cause cell damage. By lowering the melting point of water, they protect the cells which are sensitive to temperature changes and prevent the solution from forming ice crystals. Through cryoprotectants, it becomes possible to recover cells with greater functionality after storage. In order to be effective, cryoprotectants must be able to penetrate cell membranes. Examples for cryoprotectants are ethylene glycol, dimethyl sulfoxide (DMSO), glycerol or trehalose.3 In the case of DMSO, which is used to induce differentiation potential of human embryonic stem cells, undesired secondary effects such as neurological, cardiovascular and gastrointestinal side effects have been reported.4
Serum-containing medium used as a reagent for cryopreserved cell suspensions usually contain 10 % glycerol, 10 % dimethyl sulfoxide (DMSO) and cell conditioned media.5 Serum-free medium often contains up to 7.5% dimethyl sulphoxide and fresh serum-free medium with 10 % cell culture-grade. A high concentration of DMSO or glycerol should be used to function as a cryoprotectant for the cells during freezing.
Read more: Regulations for Cryoprotectants in ATMP Cryopreservation3 4 5
Next to protective agents in the solution, protective packaging is another important factor in successful cryopreservation during shipping and storage. The storage containers must be robust enough to avoid product loss, but also protected from mechanical stress, e.g. by the use of immobilizing foam. Containers like single-use bags allow for even freezing without ice crystal formations and cell degradation. Further, containers should be stored in a protective layer, which must be robust enough to endure cryogenic temperatures.
The first step of every cryopreservation protocol is the labeling and documentation of vials, bags or other containers. Adherent cell cultures have to be removed with cell culture media first and then transferred to an incubator, where they are stored for 2 minutes at 37 °C.
Afterwards, they can be filled into a tube where cell viability is determined. The required cell density is then added to a freezing medium.
The supernatant is removed and the cells can be transferred to the freezer, where they are cooled down steadily by 1 °C per minute. After 24 hours, when they have reached the desired temperature, they are stored in liquid nitrogen.6
Cryopreservation is a meticulous process that poses several challenges. Firstly, not all cells can withstand the stress of very low temperatures. Cells must be carefully selected to make sure they will survive.
Freezing rates are another important factor that influences the outcome. Temperatures should be lowered steadily as fluctuating freezing rates or an extended phase transition are due to intracellular ice formation. To prevent cells from dehydration and shrinkage, the right pace has to be found and the right cryoprotective agent has to be added to protect the cells. Some CPAs have shown to increase cell toxicity or batch variability in the interaction with specific cells, therefore the right mixture has to be chosen.
Read more about the techniques in cryogenic freezing in the article about cryopreservation techniques or how to prepare cells for the cryogenic freezing process in the article about preparation of cells before cryopreservation.
Single Use Support offers fully automated solutions for successful cryopreservation projects. As the main factors in the cryopreservation of cells are control and speed, automated fluid management and protective, scalable packaging are needed for overall process optimization.
By using a combination of single-use bags and plate freezing with RoSS.pFTU, steady and consistent freezing can be performed, reducing the probability for ice crystal formation and therefore product loss at ultra-low temperatures until -80 °C.
One of its kind, the cryogenic freezer RoSS.LN2F is the solution for controlled cryogenic freezing down to -170 C. This powerful device was developed as an enclosed and innovative platform system that helps protect frozen cells from direct exposure without the need of mechanical compressors. It ensures safe handling of cell banks with low-maintenance, high energy efficiency and process control: By injecting only as much liquid nitrogen as is necessary for a specific cooling rate, the freezing process can be carried out with enormous precision, maintaining the quality of frozen products.
Director New Products, Innovation & Product Line Management
