Introduction

Alterations in synaptic connectivity are associated with almost all brain disorders. In particular, synapse loss is a particularly serious problem in brain disorders that disrupt cognitive function, such as schizophrenia and Alzheimer's disease. Highly intrinsic, primary neuron-based screening platforms have been developed as part of a research program aimed at discovering novel mechanisms that trigger an increase in the number of neural connections as a strategy to combat these diseases, while facilitating the development of new chemical probes that trigger an increase in the number of synaptic connections. These probes will serve as a platform for the development of future generations of drugs to treat various brain diseases.

As part of a high-content screening platform, to ensure reproducible batch-to-batch results, it is essential to ensure that all cells are inoculated consistently and that cell activity is maintained for the duration of the experiment. This application note describes an experimental procedure that highlights the suitability of the MANTIS® Liquid Processor as a precise, reliable and robust solution for dispensing even the most sensitive primary neuronal cells.

Materials

1x PDL coated 1536-well Aurora microtiter plate

50 mL of medium containing .

- 1X Neurobasal-A medium
- 2.5% Glutamax-I
- 02% Gentamicin reagent
- 2% B-27 Supplements
- 10 uM FUDR

5×106 primary cortical cells extracted from P0 mouse pups

FORMULATRIX® MANTIS Liquid Processor

Eppendorf 5810 R Centrifuge

GE IN Cell Analyzer 6000 laser-based confocal imaging platform

Methods

1. Neuronal cells were inoculated on 1536-well Aurora microtiter plates
2. Dispense 15 μL of medium containing 3 x 103 cells into each of 1536 wells
3. Centrifuge the microplates at 200 g for 1 min.
4. Cells were cultured for 7 days without feeding
5. Cell counting by DIV7 Phase Contrast using IN Cell Analyzer 6000

Results

After 7 days of incubation, the mean phase cell counts were comparable to those of 96 or 384-well plates, with a CV of less than 10% for cell count uniformity across 1536-well plates.

Figure 1. Primary neuronal cells in a single well of 1536-well Aurora microtiter plate

Summary

The Formulatrix MANTIS is capable of dispensing cells consistently, even to small volumes of 1536-well plates. In addition, by using a gentle, low-shear dispensing technique, the instrument can prolong in vitro cell activity.

Acknowledgements

This work was done by Chris Hubbs of the Scripps Research Institute in Jupiter, Florida. The Scripps Research Institute (TSRI) - one of the world's largest private, nonprofit research institutions - stands at the forefront of basic biomedical science and is an essential component of medical research aimed at understanding life's most fundamental processes. Over the past several decades, the Institute has a long track record of making significant contributions to the improvement of health and the human condition.