Cell Storage

Unlike "ordinary" assay reagents (e.g., enzymes), living cells are prone to change during their time in culture, in response to environmental changes, and they require between 4 and 24 h after plating to adhere properly. Differences in cell density, metabolism and function one has to take into a consideration if using cells as reagents. Cells often function optimally at defined time points after plating or confluence [26].

For the optimal use of investment and throughput, an HTS should be performed for five to seven days each week, and consequently cell plates with functional identical cells must be available during the entire week. Preparing cell plates every day is a relatively safe solution to this problem, but it requires expensive investment in automation systems. Equipment for automated cell cultivation and plate preparation is available from The Automation Partnership (Cellmate, TSelect) and RTS Life Science International (acCellerator).

For several assays it is possible to adjust cell numbers and media conditions in order to provide functionally comparable cells for one to four days after seeding. Using this concept, cells could be delivered for HTS throughout the week, by preparing cell plates on two or three days. For other assays, however, an identical signal could not be produced at different times after plate seeding. In particular, the signals of reporter assays are known to be sensitive to changes in cell number [36].

The use of growth-arrested cells for screening provides another possibility of enhancing flexibility and reproducibility. Kunapuli et al. used CHO cells which had been frozen after mitomycin C-induced division arrest in a calcium flux assay, and reported a stable signal-to-noise ratio for four days after plating [37]. Similarly, by using mitomycin C treatment, Fursov et al. improved the robustness of a reporter assay to a Z'-value of 0.79, compared to 0.35 for normal cultured cells [36].

Differentiated cells reflect naturally growth-arrested cells by keeping their functional phenotype stable for several days in culture. Differentiated cells are more difficult to prepare, but this is acceptable if they provide reliable cell material for screening, as do Caco-2 cells [4] and differentiated 3T3-L1 adipocytes.

Maintaining plated cells under hypothermic conditions (4-8 °C) offers another possibility of enhancing flexibility in cell delivery. By using special solutions (e.g., Hypo-Thermosol; BioLife), normal coronary artery smooth cells, hepatic cells and skeletal muscle cells can be kept for between 2 and 7 days at 4-8 °C [38]. This effect is due to the solutions balancing the altered cellular ion concentrations and counteracting cell swelling at low temperature.

0 0

Post a comment