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Ensure success via a systematic cell culture workflow

21st June 2016

Posted By Paul Boughton


An overview of the cell culture workflow. Once cells are plated, health and confluency are monitored under the microscope until they are dissociated and counted in-solution, for passaging, storage or experimental use
The cell growth phase.Cell proliferation begins in lag phase, turning into the exponential log phase as growth factor concentration increases. Growth plateaus as nutrients are consumed, and increased cell density leads to contact inhibition
Modern systems enhance the cell culture workflow. Standardisation, efficiency and documentation are enabled by Olympus cell culture solutions, including the CKX53 cell culture microscope, CKX-CCSW cell confluency checker software and automated cell counter R1
Monitoring cell growth ensures a standardised process. Choosing the right time to process proliferating cells is vital, ensuring they have grown enough for a sufficient yield, but have not yet reached saturation

Jan Barghaan explains how modern technologies are bringing speed, accuracy and standardisation to cell culture

An effective cell culture process lays the foundation for success in many application areas throughout life science and the pharmaceutical industry, from cancer research to regenerative medicine. Cell biologists must consider many fundamental aspects at every stage of the workflow to ensure quality and reproducibility (Fig.1.). Although it often varies how biologists manage each task, modern laboratories are introducing approaches that ensure standardisation and accurate documentation, alongside speed and efficiency.

Standardising conditions enhances experimental success, ensuring that samples are comparable. When cells are cultured in variable conditions, this alters growth patterns and in turn is highly likely to alter the gene expression of many cellular functions.

Documenting results provides an accurate picture of cell behaviour over time, allowing traceability for future reference, audits, peer review queries or patent applications.

Speed and efficiency maintains culture health by minimising the time cells remain outside optimum incubation conditions; while fast operation also frees up time for scientists to focus on other tasks.

These aims are facilitated by a range of tools – from automated cell counters and software to specialised cell culture microscopes. In turn, the success of the cell culture process affects the reliability, reproducibility and ultimately the credibility of downstream experiments.

Observing cell cultures

Success in cell cultivation arises from insightful observation and workflow efficiency. However, traditional light microscopy systems have limitations in terms of ease of use and ergonomics, leading to a time-consuming and uncomfortable workflow that can also compromise cell culture observation. As microscopy has evolved over the years, systems specialised for cell culture observation and analysis have incorporated a number of features to overcome these challenges, from the ability to fit inside the clean bench, to novel optical techniques (Fig.2.). With the latest optics, cell culture microscopes facilitate fast and efficient screening, and feature a large field number to expand the field of view. This allows easy inspection of multiwell plates, which is ideal for cell cultures in a variety of different microplate formats.

The most popular illumination method for cell culture observation, Phase Contrast has also now been optimised. Allowing fast and high contrast observation of phase objects, a new integrated Phase Contrast (iPC) technology removes the need to individually change the ring slit when switching from 4X-40X objectives. Moreover, enhancing observation of stem cell colonies, a new contrast technique known as inversion contrast (IVC) has been developed by Olympus. This novel method extends phase contrast to generate clear, artefact-free images with enhanced 3D information, delivering a greater level of optical information, especially from thicker samples such as induced pluripotent stem (iPS) cell colonies. 

Monitoring cell growth

Cell culture growth undergoes three distinct phases (Fig.3.), and it is crucial to prevent the culture from proliferating beyond the log phase, where growth slows. In practical terms, the culture is ready to passage when confluency lies between 70-80%, and estimations are often made visually, which can be highly variable. Advances in software technology have now introduced accuracy and standardisation to confluency measurements for adherent cells (Fig.2.). Through employing such software with a cell culture microscope, quantifiable cell growth data is quickly generated to ensure cells are always passaged at the correct time (Fig.4.). Moreover, this enables scientists to create an accurate growth log, avoiding unnecessary dissociation and in-solution counting for the optimisation of culture conditions, for example.

Processing cells

Whether preparing cell samples for passaging, downstream experimentation or storage, accurate in-solution cell counts are vital:

Passaging – consistent seeding densities mean cultures grow at the same rate and health

Downstream experimentation – identical cell counts facilitate comparable results

Storing – knowing the cell concentration and viability in each vial is important when reviving cell line.

Traditionally, this count is achieved manually with the hemocytometer. However, this slow and laborious technique introduces variations due to human error. A modern alternative is automated cell counting systems, which create an accurate cell count report in just 15 seconds that can be stored and exported, for streamlining, standardising and documentation (Fig.2.).

Summary

Cell culture forms the cornerstone of life science applications, and high quality results demand a high quality cell cultivation process. This can be enabled through considering many parameters and adopting the latest laboratory technologies available. Scientists are supported in perfecting a fast workflow that is fully documented for future records, and highly standardised to achieve successful life science experiments and regenerative medicine applications.

For more information, visit www.scientistlive.com/eurolab

Jan Barghaan is with Olympus.  





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