Impact of phenol red in cell culture and solutions

What is the function of phenol red in cell culture?

Phenolsulfonphthalein, commonly referred to as phenol red, has been a standard component of cell culture media for several decades. This organic compound serves as a pH indicator, changing color in response to pH variations. Its ability to change color in response to pH variations is due to its molecular structure, which undergoes conformational changes in different pH environments.¹

At physiological pH (between pH 6.8 and 8.2), phenolsulfonphthalein exhibits its characteristic red color (Figure 1). When the medium becomes acidic (pH 6.4 or lower), it turns yellow, signaling potential bacterial contamination or excessive cellular metabolism. Conversely, a shift to purple (pH 8.0 or higher) may indicate alkaline conditions or contamination with alkaline substances.^1–3

In addition to helping researchers quickly monitor the pH of the cell culture and detect potential contaminations, the distinctive color changes of phenol red in cell culture also allow for easy monitoring of cell metabolism. This is because actively growing cells produce acidic metabolites that gradually lower the pH of the media.^2,4

A rapid change from red to yellow suggests accelerated cell growth, nutrient depletion, or waste product accumulation. This visual feedback system is a simple and effective way to monitor cell culture health and to determine when media changes are necessary.

Limitations of phenol red-containing culture media

Although phenol red in cell culture allows for quick and easy pH monitoring, its presence can influence the results of cell culture experiments in several ways:

• Estrogenic activity: It exhibits structural similarity to certain steroid hormones and possesses weak estrogenic properties that can affect hormone-sensitive cells. Therefore, the presence of phenol red in cell culture could skew results in endocrine research and studies involving xenoestrogens or breast cancer cells.^5,6
• Fluorescence interference: It can interfere with fluorescence-based assays by contributing to background fluorescence or by absorbing light at specific wavelengths.⁴ This spectral interference can affect the results of flow cytometry, fluorescence microscopy, and other fluorescence-based assays.
• Potential cytotoxicity: Phenol red is generally considered non-toxic at the concentrations used in cell culture media. However, some studies have suggested that it may have cytotoxic effects on certain cell types, potentially impacting cell growth and experimental results.⁷ The sensitivity to phenol red appears to vary among different cell types, with some cell types (e.g., breast cancer cells) showing more pronounced effects than others, adding another layer of complexity to experimental design and interpretation.⁸
• Cell-specific effects: Some studies have shown that the presence of phenol red in cell culture can influence cell proliferation rates, protein expression patterns, and cellular responses to various stimuli.^4,9,10 These effects vary among cell types and experimental conditions, adding an unwanted variable to research protocols.
• Assay interference: Phenolsulfonphthalein can affect colorimetric and spectrophotometric measurements, potentially compromising experimental accuracy.^2,11 This interference can lead to false positives or negatives, potentially affecting drug development pipelines and research findings.

Moving beyond phenol red in cell culture research

The transition to a phenol red-free environment necessitates alternative methods for monitoring culture conditions. Modern laboratories employ various strategies, including:

• Separate pH meters with specialized probes for periodic measurements¹²
• Other pH indicators with different spectral properties, such as bromothymol blue and m-Cresol purple¹³
• Advanced monitoring systems integrated with incubators¹⁴

These alternatives often provide more precise pH measurements than visual inspection of the color changes. Nevertheless, they typically require additional handling or equipment and may not be suitable for routine cell culture maintenance. Moreover, although other pH indicators can be used instead, introducing new components often requires additional optimization of culture conditions.

The clear solution: Using media without phenol red

The most straightforward and increasingly popular solution is the use of cell culture media without phenolsulfonphthalein. These formulations maintain the necessary nutrients for cell growth without the potential interference.

Advantages of phenol red-free media include:

• Elimination of potential hormone-like effects, allowing for more accurate studies of hormone-responsive pathways and improved reliability in endocrine disruption research.^4,5
• Improved assay accuracy and reproducibility, providing more consistent results across different experimental setups and enhanced sensitivity for detecting subtle cellular responses.^5,15
• Greater flexibility in experimental design, providing improved compatibility with a wide range of assays and greater control over the chemical composition of the culture environment.^4,10
• Reduced interference in optical and fluorescence-based techniques, allowing for clearer imaging of cellular structures and processes and improved performance in high-throughput screening applications.⁴
• Regulatory compliance in biopharmaceutical manufacturing, reducing the potential for unexpected interactions between media components and the final product.^5,16

Applications of phenol red-free media

Media without phenolsulfonphthalein are ideal for use in various research areas and experimental setups:

• Hormone-dependent studies, including breast cancer research and endocrine disruption investigations.⁵
• Fluorescence-based experiments, including high-resolution fluorescence microscopy, flow cytometry, high-content screening, and live cell imaging.⁴
• Response tracking studies, including drug screening, toxicology investigations, and cellular metabolism studies.⁵
• Stem cell research, including differentiation studies and regenerative medicine applications.¹⁷
• Manufacturing of biopharmaceuticals under strict regulatory guidelines, including production of recombinant proteins, manufacturing of cell-based therapies, and development of vaccines.¹⁷

These applications benefit from improved experimental accuracy, enhanced reproducibility, and reduced interference with analytical methods.

Cell culture solutions without phenol red

As cell culture experts, we recognize the growing demand for cell culture media without phenol red. We offer an extensive range of ready-to-use prf formulations designed for diverse research needs:

• Cryo-SFM Plus phenol red-free provides superior cryopreservation performance for sensitive cell types. The formulation ensures high post-thaw viability and maintains cellular characteristics during long-term storage.
• Mesenchymal Stem Cell Growth Medium XF supports robust expansion of mesenchymal stem cells while maintaining their multipotency. This specialized formulation enables maintenance of the self-renewal ability and differentiation potential of stem cells.
• Hematopoietic Progenitor Cell Expansion Medium XF facilitates optimal growth and maintenance of hematopoietic stem and progenitor cells, which are crucial for blood cell development research.
• Mesenchymal Stem Cell Growth Medium 2 ensures the robust expansion of mesenchymal stem cells from various sources and maintains their marker expression and differentiation capacity.
• Pericyte Growth Medium 2 supports optimal growth and maintenance of pericytes. This formulation preserves pericyte-specific markers and functionality.
• 3D Tumorsphere Medium XF supports the formation and growth of cancer stem cell-enriched spheroids and enables studies of tumor initiation, progression, and drug resistance. This prf version of this medium is particularly valuable for breast cancer research, where the absence of phenol red’s estrogenic properties ensures more reliable results in studying cancer stem cells and therapeutic responses.

Our commitment to research excellence extends to prf versions of media for various cell types, including endothelial cells, smooth muscle cells, preadipocytes, osteoblasts, keratinocytes, follicle dermal papilla cells, chondrocytes, airway epithelial cells, skeletal muscle cells, and myocytes. This extensive portfolio ensures that you can find the right phenol red-free solution for your specific cell type and experimental needs.

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