A comparative study of freezing single cells and spheroids: Towards a new model system for optimizing freezing protocols for cryobanking of human tumors

Fraunhofer Institut für Biomedizinische Technik, Ensheimer Strasse 48, 66386 St. Ingbert, Germany.
Cryobiology (Impact Factor: 1.59). 04/2009; 58(2):119-27. DOI: 10.1016/j.cryobiol.2008.11.005
Source: PubMed


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.

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    • "Cell culture media were obtained from Gibco (Invitrogen, Darmstadt, Germany). Spheroids were produced using hanging droplet or free spheroid technique as described elsewhere [34]. Rin-m cells and Langerhans’ islets were stained using fluorescein diacetate (FDA) and ethidium bromide [34] for viability and membrane integrity assessment. "
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    • "The intensity and rate of accumulation of the fluorescent product of FDA hydrolysis within the cells reflect both the enzymatic activity required for the hydrolysis and the integrity of the cell membrane necessary for the retention of the product. As a rule, the FDA staining is used for post-thawing estimation of cell vitality, since in the cells with damaged membranes, the accumulation of hydrolyzed fluorescein does not take place and the intracellular fluorescent signal is not observed [21,22]. Figure 5 presents a representative FDA staining image of U937 cells exposed to the freezing - thawing cycle in the i3C, at 3 hours after thawing. "
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    • "In order to determine plasma membrane integrity for the cell viability assay, cells were double-stained with PI and FDA. A mixture of FDA (final concentration 1.2 μM) and PI (final concentration 2.5 μg/ml) was added to U937 cells loaded into the i3C, for 5 minutes in the dark, at RT (the method is similar to that reported by Ehrhart et al. 2009 [33]). The cells were then imaged using fluorescence cubes for fluorescein and PI. "
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