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Accelerating Experimental Success Through a Systematic Cell Culture Workflow

The expansion and modernization of the cell culture process are driving demand for technologies that enable standardization, accurate documentation, increased speed, and reduced workloads. This white paper describes solutions that support an efficient and standardized cell culture workflow to help accelerate experimental success in cell-based applications.

Increasing Demand for an Improved Cell Culture Workflow

An effective cell culture process lays the foundation for success in applications throughout the life science and pharmaceutical industries, from stem cell and cancer research to regenerative medicine. The ability to accurately observe and document cells as they grow, proliferate, and differentiate in vitro is central to ensuring quality and reproducibility throughout the cell culture workflow. To address this, many laboratories are introducing technologies that support standardization and accurate documentation, while also improving speed and efficiency.

Supporting Efficiency, Standardization, and Documentation in Cell Culture

At its most basic, the cell culture process involves quantifying cell proliferation and density while evaluating cell morphology. Adapting to a cell culture workflow that makes these processes more efficient, accurate, and standardized helps improve the reproducibility of downstream experiments:

  • Standardizing conditions enhances experimental success, as samples are comparable. Cells cultured under inconsistent conditions can demonstrate altered growth patterns and gene expression, impacting cellular function.
  • Documenting results provides an accurate picture of cell behavior over time, enabling traceability for future reference, audits, peer review queries, or patent applications.
  • Speed and efficiency maintain culture health by minimizing the time cells are outside of optimal incubation conditions; a fast workflow also frees up time for scientists to focus on other tasks.

Modern cell culture solutions, such as an incubation monitoring system that collects quantitative data remotely or a microscope designed for cell culture needs, support this optimized workflow (Figure 1).

Figure 1. Technologies that support an optimized cell culture workflow

Figure 1. Technologies that support an optimized cell culture workflow

Hardware Features for an Improved Cell Culture Workflow

Success in cell cultivation comes from insightful observation and workflow efficiency. However, conventional light microscopy systems have limitations in terms of supporting ease of use and ergonomics. These limitations can lead to time-consuming processes that compromise cell culture observation.

As microscopy has evolved, systems designed for cell culture observation and analysis have incorporated various hardware features to address these challenges (Figure 2). 

Figure 2. The CKX53 cell culture microscope (left) and the Olympus Provi™ CM20 incubation monitoring system (right) offer features to streamline standardization and quality control in the cell culture workflow.

Figure 2. The CKX53 cell culture microscope (left) and the Olympus Provi™ CM20 incubation monitoring system (right) offer features to streamline standardization and quality control in the cell culture workflow.

For example, ergonomic components, such as natural hand positioning for focusing and sampling, increases the speed of cell processing. This benefits the cell cultures, since any time that cells spend outside of optimal incubation conditions increases the risk of contamination. Added time can also stress the cells, which can alter physiology and lead to inconsistencies between samples.

These systems also feature a compact design to fit inside biosafety cabinets. Observation can be carried out in a sterile environment, reducing the risk of contamination.

Fast and High-Contrast Observation for Cell Culture

Another improvement to the cell culture workflow is the ability to perform fast and high-contrast observation. Pairing cell culture microscopes with advanced optics provides a clear, wide view that facilitates fast and efficient screening. For instance, an objective with a field number (FN) of 22 produces images 21% larger than an objective with a FN of 20. Further, a 2X objective can provide higher contrast for a clearer sample overview. Users can easily inspect multiwell plates through one clear, large image.

Advances in observation methods also improve the imaging process for cell culture. While standard light microscopes rely on brightfield imaging, this technique has a significant limitation that makes it unsuitable for cell culture applications in many cases. Known as phase objects, live unstained cells, such as those in culture, do not absorb light. As a result, many structures are invisible under standard brightfield microscopy. Still, when light passes through these transparent samples, it undergoes a phase shift. Specialized illumination methods—including phase contrast and Hoffman modulation contrast—can transform these phase shifts into a light intensity pattern to enhance image contrast. Phase contrast, in particular, is a popular method for cell biologists. However, it requires preparing and centering each objective, and using each objective with an appropriate phase ring. This can be time consuming and lead to the incorrect phase ring being used when switching magnifications. 

To address this, Olympus offers integrated phase contrast (iPC) technology, which provides a high contrast image without the need to change the ring slit when switching between 4X–40X objectives. This simpler workflow improves efficiency and removes a potential point of inconsistency between researchers.

Enhanced Observation of Stem Cell Colonies Using Inversion Contrast

Although contrast imaging techniques are enough for standard cell culture inspection, these approaches have a limitation. Both phase contrast and Hoffman modulation contrast give rise to artifacts that interfere with the detailed observation of sample structures. Phase contrast causes a halo effect around the edge of each object obscuring its outline, while Hoffman modulation contrast introduces a shadow in a direction determined by the equipment setup. Both effects impact the precise morphological analyses required for stem cell colony cultures.

Research efforts have focused on overcoming these limitations, and a contrast technique known as inversion contrast (IVC) has been developed by Olympus. This novel method extends phase contrast illumination technology to create clear images with enhanced 3D information to deliver a greater level of optical information from a sample.

Figure 3. Inversion contrast (IVC) technology

Figure 3. Inversion contrast (IVC) technology

IVC: Extending Phase Contrast

In the unique setup of IVC, both halos and directional shadows are removed. Essentially, an annular aperture is inserted into the light path at the front focal plane of the condenser (see Figure 3). Since the aperture is larger than that used in standard phase contrast (the outer slit diameter is 10~20% larger than the pupil size and the inner slit diameter is 1~10% smaller than the pupil size), after passing through the sample, the beam illuminates the edge of the objective’s pupil.

Therefore, only light passing through the sample at a small angle passes through the objective, while light at a large angle does not. When the sample has phase variation, the direction of the light passing through the sample changes. As a result, the image of the annular aperture is shifted slightly from the position of the pupil of the objective, and the area of the light flux passing through the objective increases.

The increased light forms an image devoid of halos. With the concentric arrangement of annular aperture and objective, the setup is independent of phase shift direction, eliminating directional shadows. The contrast is inverted across the focal plane, and the depth of field is educed.

The IVC method significantly facilitates the morphological analysis of 3D cells, such as induced pluripotent stem cell (iPS) colonies. By clearly observing iPS colonies, it is possible to detect changes in morphology suggesting cellular health or differentiation. 

To demonstrate its value, the IVC method was applied to visualize the outline and structure of mouse iPS cells (iPS-MEF-Ng-20D-17, Kyoto University), capturing the 3D nature of the cells when compared to phase contrast. IVC has also been found to be ideal for distinguishing iPS cells from the surrounding feeder cells. The contrast of the feeder cells is lower due to their flatter morphology, highlighting the iPS cells against the background.

Figure 4. Mouse iPS cell colonies documented with (a) inversion contrast (IVC) compared to (b) standard phase contrast. The IVC technique provides enhanced 3D information (c).

Figure 4. Mouse iPS cell colonies documented with (a) inversion contrast (IVC) compared to (b) standard phase contrast. The IVC technique provides enhanced 3D information (c).

Versatility for a Wide Range of Observation and Analysis Tasks

Extending the versatility of the cell culture microscope, fluorescence imaging capabilities enable the researcher to perform routine observation and functional studies on the same system. This saves both space and costs. A single system can be used for multiple stages of the cell culture workflow: from monitoring the seeding, proliferation, and passaging to high-contrast fluorescence imaging of cellular assays using a wide range of dyes.

Generating Quantifiable Cell Growth Data

An essential piece of successful cell culture is the proper monitoring of cell growth and confluency. Cell culture growth undergoes three distinct phases: lag phase, log phase, and plateau phase (Figure 5).

Figure 5. Cell growth

Figure 5. Cell growth

Cell proliferation begins in the lag phase, turning into the exponential log phase as the growth factor concentration increases. When nutrients are consumed or cell density increases, contact inhibition occurs and cell proliferation stagnates. Choosing the right time to process proliferating cells is vital since it helps ensure that they have grown enough for a sufficient yield but have not yet reached saturation.

It is crucial to prevent the culture from proliferating beyond the log phase, where growth slows. In practical terms, the culture is usually ready to passage when confluency reaches 70–80%. Estimations of confluency are often made visually, which can be highly variable. Advanced imaging software is now available that can introduce accuracy and standardization to confluency measurements for adherent cells (Figure 2).

By employing this software with a cell culture microscope, scientists can quickly generate quantifiable cell growth data to check that cells are passaged at the correct time (Figure 6). Further, this capability enables scientists to create an accurate growth log, avoiding unnecessary dissociation and in-solution counting for the optimization of culture conditions.

Figure 6. Monitoring cell growth enables a standardized process

Figure 6. Monitoring cell growth enables a standardized process

A Systematic Approach to Cell Counting

Automated cell culture systems can improve the accuracy and speed of cell counting. Whether preparing cell samples for passaging, downstream experimentation, or storage, accurate cell counting is critical for the following reasons:

  • 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 a cell line from storage

Traditionally, cells are counted manually with the hemocytometer. However, this slow and laborious technique introduces variability due to human error. An alternative method is an incubation monitoring system (Figure 7) that can automatically acquire quantitative data while the sample is left in the incubator.

Figure 7. The CM20 incubation monitoring system acquires cell data in the incubator

Figure 7. The CM20 incubation monitoring system acquires cell data in the incubator

This system makes it possible to acquire continuous quantitative data while minimizing damage to cells. It also eliminates the need for workers to perform an enormous amount of routine measurement work.

Summary

Cell culture forms the cornerstone of life science applications, and high-quality results demand a high-quality cell cultivation process. By optimizing optical methods and adopting smart laboratory technologies, scientists can create a cell culture workflow that is efficient, fully documented, and highly standardized. As a result, scientists can accelerate their work for life science experiments and regenerative medicine applications.

Author

Joanna Hawryluk, Product manager
Olympus Corporation of the Americas 

References

  1. F. A. Ross, Phase Contrast and Interference Microscopy for Cell Biologists (Edward Arnold, 1967)
  2. R. Hoffman, J. Microsc. 110, 205 (1977)
  3. Suzuki, Y., Kajitani, K. and Ohde, H. (2015). Method for observing phase objects without halos or directional shadows. Optics letters; Vol. 40, No. 5. pp 812-815
  4. K. Okita, T. Ichisaka, and S. Yamanaka, Nature 448, 313 (2007)

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