Selection of the Most Suitable Cell Line

A selected cell line for HTS (Fig. 5.1) must express the desired function in the correct signal context with a stable and reproducible signal window.

The different cell types from which to choose have different advantages: primary cells best resemble the physiological situation [3], but cultivation and available cell numbers are critical issues, even when using techniques such as conditional immortalization [4] or stem cell differentiation.

A large panel of easy-to-cultivate immortalized cell lines is available with cell history, growth behavior and functional information [5-8]. These cells are ideal for functional screens as proliferation assays.

Fig. 5.1 From cell selection to plate production. The diagram shows the different work packages with the most important parameters for analysis and optimization.
Table 5.1 The generation of recombinant cell lines.

Stable cell lines

Reproducible material with selected expression level

Frequently used • Nonviral: electroporation plasmid delivery (mainly suspension cells) methods: • Polyethylenimine (PEI), calcium phosphate, liposomal reagents • Retroviruses (e.g., lentiviruses)

Plasmid integration • Random gene expression ± methods: amplification; high producer

• Targeted expression as Flip-in system; fast cell generation

• Random gene activation; work-intensive but no intellectual property

Transient bulk transfection

Reproducible if portions of one batch are produced

Toxic proteins can be expressed, no cell clone selection

Timeframe can be shorter as generation of stable clone

Delivery methods: • Developed method for few cells as 293 EBNA with inexpensive reagents as PEI and calcium phosphate • Infection with adenoviruses and Semliki Forest virus

Transient trans-fection of every well

Reproducibility often a problem without automation

On-time delivery for HTS is not easy without automation

Recombinant cell lines overexpressing the target are often used for screening, because cell lines with an optimal growth behavior and signal window can be selected (Table 5.1). In order to obtain reproducible cell material for HTS, the favorite procedure is to generate a stable target-expressing cell line. This allows adjustment of the expression level by using different promoter elements and/or amplification with the methotrexate or glutamine synthase [9, 10] system combined with clone selection. Several transfection methods are available [11, 12] as well as infection with retroviral vectors [13, 14].

Targeting expression systems such as the Flip-in system (Invitrogen) [15] can help to shorten cell line development time to between one and three months, and if the target is restricted by intellectual property or the generation ofa recombinant cell is complex, then the random gene activation strategy can be considered [16]. Before HTS can be carried out, a master cell bank must be prepared and the stability of the functional target expression should be tested for a period of more than four weeks.

In addition to stable cell lines, identical cell material can be produced by transient transfection of one large cell batch, followed by freezing the transfected cells in portions. Directly before HTS, the frozen cells are seeded into assay plates. For an optimal large-scale transient transfection, special standards must be fulfilled for the cell line, the expression plasmid, the medium, and the transfection reagent

[12, 17, 18]. By using inexpensive reagents such as PEI [19] or calcium phosphate [20, 21], cells could be reproducible transfected in suspension culture [21-23], roller culture or cell factories (Nunc). An alternative is to infect the cells with Semliki Forest virus (SFV) vectors [24] or adenoviruses (QBiogen) with a broad host range.

The transient transfection of every single well of an assay plate requires a long development time, is work-intensive, and tends to be less reproducible, though it can be improved by using automated cell cultivation and plating systems [25].

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