University of Heidelberg
BIOQUANT

Multi-Scale Imaging & Data Mining

First prototype of a microwell cell array
Cell array containing 9216 physically separated microwells. The arrow (yellow, small figure) depicts reference markers.

 

Novel probe substrates and printing technology for High-Content Screening

 

High-Density Microwell Cell Arrays

 

Reverse transfection on cell arrays is a high throughput method for parallel transfection of mammalian cells. GFP tagged cDNAs or small interfering siRNAs are printed together with a transfection solution at defined locations (spots) on glass slides resulting in “ready to transfect” substrates (solid phase transfection; see Automated Sample Preparation). The dried and “ready to transfect” replicates can be stored for more than 15 months.

Disadvantages of the standard “array” reverse transfection technique are the risk for cross contamination and the lack of reference markers for fully automated screening pipelines. A big drawback of the well based transfection is the limited number of samples (96, 384 or 1536).

For this, we develop microwell cell arrays * overcoming the problem of cross contamination by using physically separated cavities on glass slides by a titanium coating. The dimension of the cell array with the connected polystyrene frame for cell solution handling fits the SBS (Society for Biomolecular Screening) plate standards (see Figure, top right).

 

* Reymann J. et al. (2009), Next generation 9216 microwell cell arrays for high content screening microscopy, BioTechniques

Feasibility of the microwell cell array: A very high transfection efficiency of solid phase reverse transfection by phenotypic penetrance could be proven without observing cross contamination between neighbouring spots at the same time (see Figure, below).

Figures 2B – 2G depict the specificity of samples with the target specific primers INCENP and PLK as well as samples with a non silencing unspecific control siRNA. The here used HeLa cells show the expected characteristics for the corresponding transfection with siRNA: INCENP resulted in multilobing of the cell nuclei due to segregation defects (Figure 2E) and knocking down of PLK clearly caused mitotic phenotypes of prometaphase arrest with a very high transfection efficiency (Figure 2G). In contrast, the non silencing control siRNA showed no effects (Figure 2C).

 


"whole genome" Microwell Cell Arrays

 

On-going improvements address the optimisation of the cell array in terms of a

 

  • Reduction of the thickness of the glass slide (down to 170µm) to access high resolution microscope setups
  • Further increase of the spot number by reducing the spot-to-spot distance
  • Improvement of the coating for minimising cell growth in between the cavities

 

 

Contact: E-Mail (Last update: 07/06/2012)