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Introduction
In recent years cell culture has played an increasingly importantrole in high-throughput screening applications. The impact ofcell culture data on drug discovery and subsequently on drugdevelopment to the final product is highly significant.

An essential requirement for culturing cells in vitro is a surfacesubstrate which provides a basis for firm attachment ofanchorage-dependent cells under in vitro culture conditions.Besides physically treated culture vessels, surfaces coatedwith biomolecules are an additionally powerful tool for theseapplications. In high-throughput screening (HTS) the need forautomation often results in experimental setups which arestressful for the cells. These applications require a more effectivesurface for cell attachment. High injection pressures of buffersduring washing steps are often unavoidable and may lead tosignificant loss of cells using standard tissue culture treatedvessels and classic culture conditions. Therefore, cell cultureexperiments using non-coated surfaces for cultivation aresometimes not feasible and have to be replaced by proteincoated culture vessels.

Greiner Bio-One offers coated surfaces with natural proteinsand synthetic polypeptides like Collagen Type I and Poly-Lysine for the cultivation of specific cell lines and variousexperimental procedures. Poly-Lysine enhances the attachmentof cells by generating a positive charge on the surface. Theapplications in Poly-Lysine coated culture vessels include theintegration of washing steps in an experimental setup, thecultivation of cells under serum-free or serum-reducedconditions¹ or the transfection of cells. It has been proventhat Poly-D-Lysine (PDL)-coated surfaces provide beneficialadvantages when culturing neuronal cell lines with respect toboth viability and proliferation.

Two forms of Poly-Lysine, Poly-L-Lysine (PLL) and Poly-DLysine(PDL) which differ in the steric configuration of themolecule, are available on the market (Fig. 1). These moleculesare the mirror image of each other, termed enantiomers. Whileboth coatings are widely used, PDL unlike PLL is notdegraded by cellular proteases. Therefore, it may be thepreferred coating for some applications and cell lines.

As a synthetic protein, Poly-Lysine increases cell attachmentwithout influencing specific signalling pathways in the cell.Therefore, the numbers of different parameters influencing thecells are minimised and more specific conclusions on thedrugs under test may be obtained. Another advantage of thisspecific coating is that Poly-Lysine is a synthetically producedpolypeptide and as such cannot introduce any animal derivedimpurities into the cell culture system.

Greiner Bio-One also offers culture vessels coated withCollagen Type I. Collagen Type I is one of the major proteinsfound in the extracellular matrix and in connective tissue likeskin, tendon and bone. In vivo, the cellular interaction withCollagen Type I is mediated largely through integrin receptors². In vitro, Collagen Type I influences the differentiation,morphology³ and growth of a variety of cell lines includingendothelial⁴, epithelial, hepatocytes and muscle cells as wellas other transfected cell lines.

The following application will focus on the impact of washingsteps in an experimental setup by using cell culture treatedand PDL-coated (CELLCOAT^{/sup>) microplates.}

Material and Methods

Cell culture
Using an MTT assay, the effect of washing steps on cellattachment is demonstrated in the following experiment. TheMTT assay is a laboratory test for measuring cell vitality bymitochondrial activity. Yellow MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide) is reduced to purpleFormazan by a mitochondrial reductase (Fig. 2).

The amount of purple Formazan formed is indicative of thevitality of the cells. The Formazan is dissolved in acidic Isopropanolafterwards and then quantified by measuring theabsorption at 590 nm.

To demonstrate the effect of washing steps on cell attachment,a Neuroblastoma cell line was seeded on cell culturetreated and PDL-coated microplates in densities of 50,000cells/well; 100,000 cells/well and 200,000 cells/well.

DMEM medium supplemented with 20 % FCS and 1 %Penicillin-Streptomycin was used for cultivation.

After an incubation of 45 min at 37C and 5 % CO₂, thesupernatant was discarded by aspiration. Then, the cells werewashed by pipetting. The washing solution was aspirated andreplaced by medium containing MTT in a concentration of1 mg/ml. The microplates were incubated for 1 hour at 37Cand 5 % CO₂. Pictures were taken before the washing stepand after adding the MTT.

Finally, the Formazan formed by living cells was dissolved in150 acidic Isopropanol and evaluated by measuring theoptical density at 590 nm.

Results
Microscopical observation
In order to determine the optimal cell concentration, variouscell densities were seeded in 96 well microplates in triplets.Cell densities of 50,000 cells/well; (Fig. 3) 100,000 cells/well (Fig. 4) and 200,000 cells/well were used (Fig. 5).

After incubation of one hour at 37C and 5 % CO₂, cells weremicroscopically observed. The attachment of the cells on thePDL-coated microplates is significantly better than on the cellculture treated microplates. On the PDL-coated microplates,cells had already started to spread out and showed the firstsigns of adherence (Fig. 3a, 4a and 5a). In contrast, cells onthe cell culture treated surface had a round morphology andformed clusters of floating cells (Fig. 3c, 4c and 5c).

To demonstrate the impact of specific experimental conditions,like washing steps in automated systems, the cells werewashed several times by pipetting. On the cell culture treatedsurfaces nearly all cells were washed away. Only a few cellsremained as floating clusters on the surface (Fig. 3d, 4d and 5d).Compared to these observations, the cells cultivated on PDLcoatedsurfaces attach firmly to the surface and remain completelyon the microplate bottom even after extensive washingof the wells (Fig. 3b, 4b and 5b). These observations werereproducible for all seeded cell densities and pipetted triplets.

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