A comparative study of freezing single cells and spheroids: towards a new model system for optimizing freezing protocols for cryobanking of human tumours.

Cryopreservation of human tumour cells and tissue is a valuable tool for retrospective analysis and for the transport and handling of biopsy material. Tumour tissue consists of different cell types, which have different optimal freezing conditions, and extracellular matrix. A well-defined and authentic model system is required for developing new freezing protocols and media. This work describes the use of L929 and PC-3 spheroids as new model systems for freezing human tumours. Cell suspension and spheroids were frozen in different vessels (1 ml cryovials and a special, cryo-compatible 30 x 25 microl multi well plate) at slow rate (1 degrees C/min). Freezing media were combinations of culture or tumour transport medium (Liforlab) with the cryoprotective agents, Me(2)SO, trehalose and modified starch. We also present a new method of evaluating the viability of three dimensional multicellular systems to compare thawed spheroids objectively. Best viability (70%) of L929 spheroids occurred with a combination of Liforlab and starch hydrolysis product. The best cryopreservation results for spheroids were found with extracellular cryoprotectants, while optimum viability of single cells was achieved with Me(2)SO.