Cryopreservation of plant cell cultures - Diverse practices and protocols

Plant cell cultures can be used as biotechnological platforms for the commercial production of small-molecule active ingredients and recombinant proteins, such as biopharmaceuticals. This requires the cryopreservation of well-characterized cell lines as master cell banks from which uniform working cell banks can be derived to ensure high batch-to-batch reproducibility during production campaigns. However, the cryopreservation of plant cells is challenging due to their low viability and poor regrowth after thawing. Three approaches have been developed: slow freezing, vitrification, and encapsulation-dehydration. Typically, the protocols are iteratively adapted to accommodate the properties of different plant cell lines, taking time and resources while achieving moderate success. Since standardized processes are a prerequisite for industrial applications, this review presents an in-depth analysis of the different procedures for cryopreservation of plant suspension cell cultures, highlighting relevant parameters for effective cryopreservation and the re-establishment of vigorous plant cell cultures within weeks. The protocol variants are grouped into modules that facilitate the directed improvement of each step and allow protocol evolution by module recombination. Ultimately, such improved cryopreservation protocols will form the basis of processes that comply with good manufacturing practice and attract major biopharmaceutical companies to the benefits of plant molecular farming.