How Automation and Optimization Help us do Better Science

By Sylvia Dyballa, Head of Technology Development at ZeClinics.

Sylvia studied Biochemistry at the University of Tübingen (Germany). She then became interested in cellular systems and how cells behave to form tissues and organisms. So, after graduation she moved to Barcelona to pursue her PhD in Developmental Biology with Cristina Pujades at the UPF. During her PhD she started working with zebrafish, and she used live imaging and cell lineage reconstruction to generate a “digital” zebrafish organ. After her PhD, she teamed up with ZeClinics to contribute with her experience in live imaging, image- and data-processing. Today she is Head of Technology and Development in ZeClinics. Her aim is to push innovation and growth in ZeClinics through the use of emerging technologies.

In the previous article of this blog, we explored what makes zebrafish the perfect organism for imaging-based phenotypic screening. In this article, we want to focus on the benefits of optimization & automation to accelerate screening throughput and extract meaningful phenotypic features. 

To recapitulate, the key features that make the zebrafish larva such a valuable model in this endeavor are:

  • Zebrafish larvae are small in size 
  • Which means they fit well under the microscope
  • Zebrafish larvae are optically transparent 
  • Transgenic zebrafish lines are available to fluorescently mark a plethora of different cell types
  • From a single couple you obtain about 300 larvae per spawn
 

Those features allow conducting image-based assays in vivo and in a HTS (high throughput screening) manner. Indeed, at ZeClinics we develop such assays to understand and quantify developmental toxicity, specific organ toxicity and the efficacy of drugs in a growing number of disease-based screenings. What is more relevant, we have increased the throughput of those assays about 20-fold from when we started in 2013. How did we achieve that?

On the one hand, we have bet on emerging technologies to enhance our experimental work productivity – microfluidics, robotics, video-tracking, deep learning for image analysis, etc. Often, these technologies are ready for being used in a high-throughput setup, but other times additional development is needed to truly employ them in our experimental pipelines. On the other hand, we automate all phases of the experimental pipeline as much as we can, from planning to experiment, to analysis.

In ZeClinics we absolutely love this combination of challenges, because it allows us to satisfy our desire to try new things, explore new grounds, do R&D – in short: be scientists. But we are also keen to scale our solutions in order to solve real world problems. So in the development of new assays there are really two phases: first we develop the assay and validate it; next we automate the assay in order to use it in HTS.

 

Automating an assay has several advantages to it

  • A minimum of experimenter intervention is required.
  • Thus, mistakes made by humans can be largely avoided. And if they are made, since the process is fully traceable, they can be quickly detected and corrected. 
  • Variability from manual manipulation of living samples is decreased. That is, automated assays are more reproducible
  • Automated assays are much quicker than any human can do the tasks. And this allows to save vast amounts of time when conducting an assay.
  • In line with this, increased amounts of data can be obtained in a single experiment and the data is of superior quality when compared to low throughput or manual assays. 
  • This allows to also streamline the analysis of the data: since the analysis is also automated, more parameters can be analyzed and the biological insight gained from a single specimen increases.
 

Here are two assays that have benefited from automation

1. DevTox

The zebrafish larva has become quite an established model in toxicity. One can predict compound induced human teratogenicity (developmental toxicity) with high specificity (Jarque et al., 2020). To render the model suitable for HTS, ZeClinics has invested a lot of effort to streamline all parts of the procedure, from planning to experimental execution to image and data analysis. At the end of the day, that allows us to offer developmental toxicity screening as a competitive service to our customers and to easily integrate the developmental toxicity setup to any other setup (genetic mutant screens, specific organ toxicity etc.).

 

2. CardioTox

In drug discovery, cardiotoxicity is among the leading causes of failure of compounds. This is true for both, failure at late stages of drug development and – what is worse – for withdrawals after their market release. In order to detect cardiotoxicity of compounds early and reliably, we have devised the ZeCardio screening platform, which streamlines the analysis of effects of compounds on heart function in vivo. The platform is actually the combination of a robotic imaging system (VAST Bioimager), and a custom made software (ZeCardio) that we developed as part of an SME Instrument Phase 2 grant that we obtained a few years back. In order to provide this pipeline as a service to our customers, we had to put a lot of effort into optimization, standardization and automation. We have validated the platform in a compound screen of ~100 molecules with known cardiotoxicity in humans (Dyballa et al., 2019), showing that the platform, and the zebrafish larvae model, are as predictive for estimating cardiotoxicity as the gold standard model for that purpose, the dog. The effort we have put into developing this pipeline allows us to provide it as a competitive service to our customers and to integrate it into our own drug discovery and R&D projects. Indeed, the ZeCardio platform has been the experimental basis from which we have built our spin out company, ZeCardio Therapeutics.

 

In conclusion, the zebrafish larva is an excellent model for conducting HTS experiments. In the future the model is likely going to become even more powerful since optimization and automation now allow us to combine different HTS assays: we can combine compound screens, genetic manipulations, phenotypic screens, behavioral screens and transcriptomic analyses and obtain more endpoints from a single specimen. This approach should ultimately allow us to i) basically understand “disease” better and to ii) increase even further the predictability of the zebrafish model for human disease.

 

REFERENCES:

[1] S. Jarque, M. Rubio-Brotons, J. Ibarra, V. Ordoñez, S. Dyballa, R. Miñana, J. Terriente, Morphometric analysis of developing zebrafish embryos allows predicting teratogenicity modes of action in higher vertebrates, Reprod. Toxicol. 96 (2020) 337–348. https://doi.org/10.1016/j.reprotox.2020.08.004.

[2] S. Dyballa, R. Miñana, M. Rubio-Brotons, C. Cornet, T. Pederzani, G. Escaramis, R. Garcia-Serna, J. Mestres, J. Terriente, Comparison of Zebrafish Larvae and hiPSC Cardiomyocytes for Predicting Drug-Induced Cardiotoxicity in Humans, Toxicol. Sci. 171 (2019) 283–295. https://doi.org/10.1093/toxsci/kfz165.

[3] S. Jarque, J. Ibarra, M. Rubio-Brotons, J. García-Fernández, J. Terriente, Multiplex analysis platform for endocrine disruption prediction using zebrafish, Int. J. Mol. Sci. 20 (2019). https://doi.org/10.3390/ijms20071739.

 

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