Stand up and be counted

The CFC assay and colony counting is universally recognised as the gold standard for measuring the effects of radiation, chemotherapeutic drugs and other agents on cell viability. However, manually counting the resulting cell colonies is a tedious and laborious task, now however automated colony counting is opening up new possibilities for cancer biologists Highly sensitive and biologically relevant, the Colony Forming Cell (CFC) assay has a valuable place in the cancer research and drug development process. The use of highly stringent tests such as CFC assays can help to eliminate late drug failure and evaluate the viability of therapeutic combinations or dosing regimes. All of which can significantly reduce the time and cost associated with pharma discovery, drug development and the evaluation of potential new treatment methods where modes of action need to be validated accurately against oncology targets. Despite the obvious value and utility of CFC assays, the assay is generally perceived as time consuming and technically challenging to set up and analyse. The manual counting of cell colonies is a time consuming and painstaking task in which consistent objectivity is difficult to achieve. What’s more, the manual counting of CFC assays represents a significant undertaking in terms of resources and time for biopharma organisations and research facilities of all sizes. The manual counting scenario is challenging enough in adherent mono-layer ‘two dimensional’ samples. But in recent years sphere formation (or tumour sphere) assays, which simulate tissue state replication, have become increasingly popular with cancer biologists. Involving three dimensional in vitro culture systems, accurate visual recognition and consistent manual counting is more time consuming and difficult for observers to achieve. Yet the increasing emphasis on combination treatments for cancers, and the rapidly growing number of agents under development, means that today’s laboratories working at the cutting edge of research need to be able to perform with ease quantitative 3D assays that fully reflect drug and radiation combinations on proliferating tumour cells. The concept of computer-aided colony counting is not a new one. But the exponential growth of desktop computer processing power has made it cheaper, more accessible and far easier to utilise off-the-shelf computers in a wide range of medical research applications. Today’s automated colony counting and integrated image analysis systems now offer an efficient and cost effective alternative to manual counting, putting the analysis capabilities required to undertake today’s highly sophisticated 3D arrays within reach of any research laboratory. Sophisticated and robust image-processing algorithms automate the detection, counting and analysis of mammalian cell colonies in Petri dishes, flask and multi-well plates. Using high depth-of-field imaging, colonies can be imaged, processed and characterised in a single integrated hardware/software platform, eliminating the disruptive requirement for multiple devices at every stage of the process that characterised the early days of automated counting. Offering a cost effective and reliable alternative to manual counting, today’s ‘all-in-one’ dedicated colony counters deliver superior accuracy and precision in comparison to manual observer counting. Impressive colony detection performance includes resolution of the overlapping colonies challenge alongside the exact differentiation of colonies from debris or other artefacts. Offering a standardised method for automated CFC analysis, these systems at last make it possible for research labs to increase throughput and reduce workflow demands while simultaneously upping the consistency and accuracy of their results. The benefits don’t just end there. Modern intelligent systems enable operators to set defined colony thresholds for counts – for example, researchers can elect to exclude colonies based on size or colony shape-related parameters or assign general object detection sensitivity parameters. They also allow researchers to undertake repetitive plate counting of non-adherent colonies without staining cells. Technological advancements mean contemporary single instrument automated systems also provide extensive analysis parameters that make unique new insights possible and give research laboratories the facility to capture and report additional qualitative and quantitative data. Alongside generating counts, researchers processing colony samples can now collate detailed colony size information in the form of a mean-per-well/dish, histogram distribution or on an individual colony basis. This new found ability to quantitatively measure the effects of anti-cancer therapeutic regimes on absolute colony numbers and colony size makes it possible for research laboratories to extend the sensitivity of the colony forming assay and obtain previously ‘hidden’ information relating to colony growth dynamics. But today’s automated intelligence-based systems also open the way to highly sophisticated data capture and export. Full digital image archiving and per-colony raw data exportation means that images of colony plates or dishes can be output directly into lab documentation or saved as a digital raw image that supports the further processing or reprocessing of samples.

Laboratories looking to engage in multi-site research projects now have the capacity to store and reapply defined assay count parameters in order to maximise counting proportionality and reproducibility

As a result research laboratories are able to instantly ‘visually document’ individual assay findings, submitting these images to multiple teams or external groups for independent validation and/or independent processing and assessment. This new found ability to capture, process and export data makes new collaborative research approaches possible – for example, laboratories can submit assay images for rigorous double blind tests – and eliminates the challenges inherent in duplicating assay methodology when undertaking, parallel, global or large scale trials. For example, laboratories looking to engage in multi-site research projects now have the capacity to store and reapply defined assay count parameters in order to maximise counting proportionality and reproducibility. Settings can also be stored and submitted to other teams or personnel – regardless of location – making parallel validation programmes possible or enabling multiple new drug combinations to undergo parallel assessment. In a similar way, reporting (both during and post-trial) becomes a simplified and automated process; statistical distributions and summary data – including colony numbers and attributes – can be instantly exported and in a variety of formats. The arrival of modern highly sophisticated arrays means research laboratories are increasingly finding traditional manual counting approaches are cost and resource inhibitive and limiting when it comes to the precise study of drug effects with respect to both dose and time of exposure using fewer culture plates. CFC assays as an in vitro method provide a powerful tool to monitor the effects of chemotherapeutic compounds but significantly increase the complexity and time involved when it comes to manual analysis. With the increasing emphasis on combination treatment for cancers and the increasing number of agents under development means that the volumes of assays are set to grow, making reproducible and easy-to-form quantitative assays that fully reflect drug effect on both colony size and number a ‘must have’. Relying on staining and the visual recognition of colonies is no longer sufficiently precise or practical for testing. Automating the detection, counting and analysis of mammalian cell colonies offers significant benefits to cancer biologists processing tumour colony forming assays – eliminating the risks of subjectivity, bias and human error, increasing speed and accuracy, and delivering unprecedented data archiving and retrieval capabilities. Furthermore, the added benefit of new qualitative and quantitative imaging parameter analysis provides an additional layer of sensitivity to assays, allowing researchers to pick out the more subtle effects of potential treatments. Finally, the ability to capture and distribute findings and/or share the parameter settings provided by today’s integrated automated colony counting platforms make it possible for laboratories to replicate with ease large scale assays or distribute elements of large research projects. As a result, teams in multiple locations can undertake parallel assays, instantly replicating count parameters or subjecting arrays to sophisticated validation or peer-to-peer review. This new versatility ushers in a new era where laboratories are able to maximise research funding in pursuit of outcomes, employ new sensitive data collection parameters and undertake ‘industrial scale’ array counts. It also enables researchers to engage in ‘open innovation’ approaches to R&D and drug discovery where the collective expertise of a network of contributors can be harnessed with ease. Author Andy Obeid, Founder and CEO, Oxford Optronix, Contact info@oxford-optronix.com