Whilst light microscopy has long been central to process characterization, the scaling up of cell and gene therapy manufacturing is now calling for innovative alternatives in cell monitoring.
Light microscopy is not the only way scientists have been monitoring cells and more sophisticated sensors are emerging with every year. © Green Elephant Biotech.
Innovation often starts with the unconventional. Technologies that once seemed radical – from GPS navigation to neural networks, have become essential to our daily lives. The same transformation is now unfolding in life science laboratories, where cell culture microscopy is no longer the sole method for assessing cell viability and growth. In modern Cell and Gene Therapy (CGT) workflows, alternative cultivation systems are emerging, with novel monitoring techniques moving beyond traditional microscopy without compromising data quality.
These systems enable better process characterization, particularly in formats where microscopy access is limited or impractical, such as due to the scale. As demand for CGTs and early-phase biologics continues to rise, adherent cell manufacturing remains a key bottleneck. One promising solution lies in the adoption of scalable bioreactor systems, designed to standardize and automate cell growth while reducing manual variability (1). Though well established in large-scale production, bioreactors remain an investment that predisposes to the use of low-impact (i.e., without cell passaging), real-time monitoring of mammalian cell cultures.
This article explores evidence-based analytical approaches that complement or possibly replace conventional microscopy in assessing cell viability, growth, and even quality. By focusing on the methods applicable to different experimental designs, we aim to highlight how technologies ranging from metabolic sensors to optical and antibody (Ab)-based assays reshape the way cell cultures are monitored.
pH and lactate reveal more than the single-cell observations
By their nature, metabolic sensors only require low-impact sampling and are widely applied to ensure reproducibility in large-scale bioprocesses through standardized monitoring. Among the well-understood metabolic indicators, pH remains a central parameter. The concentration of hydrogen ions in the cellular microenvironment directly reflects metabolic activity and strongly influences cell physiology.
For mammalian cell culture, optimal proliferation typically occurs within a slightly alkaline range of pH < 7.0-7.4 > pH. Meanwhile, deviations toward pH > 7.4 can significantly affect gene expression, protein synthesis, and overall cell proliferation. In culture systems, pH often rises with higher cell density, as glycolytic activity leads to metabolite and lactate accumulation. These metabolic shifts alter cellular microenvironment, underscoring pH as both a driver and a reporter of cell state (2). Modern pH-sensitive electrodes and optical sensors enable continuous readouts, which can serve as soft-sensor proxies for viable cell growth, assuming metabolic activity per cell remains relatively stable (3).
This principle also extends to lactate monitoring, a critical by-product of glycolytic metabolism. When primarily consumed through anaerobic glycolysis, glucose is broken down to pyruvate, which gets processed further into lactate to regenerate NAD+ and ATP. During the exponential growth phase, lactate accumulates and cells may utilize lactate as a carbon source following the peak density.
Consequently, the interplay between glucose consumption and lactate production provides predictive insights into cell growth dynamics (4). Real-time lactate sensors, based on electrode or fluorescence technologies, underscore this analysis. Finally, similar considerations can be applied to the use of dissolved oxygen (O₂) and carbon dioxide (CO₂) due to their roles in cellular respiration and pH regulation, though their relevance in quantifying the latter is disputable.
Absorbance more precise than the “naked eye”
Further insights into cell growth monitoring can be achieved through wavelength-based analysis of culture samples. One of the most widely recognized techniques is the measurement of optical density (OD), which relies on the ability of mammalian cells to absorb and scatter light (3). A study from 2020 assessing the concept in anchorage-dependent cells has demonstrated that light absorbance can serve as an indirect measure of cell count in its proof-of-concept experiments. By establishing a linear correlation between detectable absorbance and actual cell numbers, researchers proposed using OD as a proxy for cell density. This approach still bears limitations, as at very high or non-linear cell densities, light scattering effects reduce accuracy and can lead to significant counting errors (5).
Despite these challenges, real-time optical probes integrated into bioreactors provide continuous density readouts while minimizing contamination risks associated with manual aliquot sampling or microscopy. For greater precision and biological relevancy, flow cytometry remains a gold-standard technique. Unlike OD analysis, flow cytometry enables cell cycle profiling, viability assessment, and apoptosis detection using fluorescent dyes coupled with laser-based detection. For example, markers such as propidium iodide (PI) and 7-AAD allow the quantification of sub-populations of cells in distinct phases, including the G2 phase following DNA replication and prior to mitosis (6).
In fact, automated flow cytometry protocols have already been implemented in bioreactor cultures under aseptic conditions, producing results that closely mirror conventional manual analyses. While most monitoring platforms still require careful interpretation of ambiguous results (7), the labor reduction and enhanced reproducibility benefits of flow cytometry offer valuable opportunities for scalable bioprocessing in mammalian cell culture.
One quintillion ways to monitor the cells
Ab-based biosensors, also known as immunosensors, represent a versatile class of analytical devices that integrate a biological recognition element with a signal transducer. Rather than quantifying a single physicochemical parameter, these systems detect specific biomolecular interactions and translate them into measurable digital outputs. Over the years, numerous biosensor formats have been developed for medical diagnostics, environmental monitoring, and agricultural analysis, with their sensitivity largely defined by the transducer design and the remarkable diversity of Ab available for production (estimated at up to 10¹⁸ unique variants) (8,9).
In conventional setups, immunosensors rely on a fixed substrate (e.g., silicon, glass, or polymer) coated with Ab to capture metabolites or growth factors secreted during cell proliferation. This approach is well established in clinical and bioprocessing applications, enabling the detection of disease markers or production-related analytes in patient or culture samples. Recent advances in comparison are redefining this principle by introducing mobile sensing elements, such as Ab-coated microbeads, designed to overcome the saturation and regeneration challenges inherent to fixed-surface systems.
For instance, Son et al. demonstrated a microfluidic platform employing Ab-functionalized microbeads to monitor hepatocyte growth factor (HGF) and transforming growth factor (TGF)-β1 secreted by hepatocytes. Though this model was aimed to determine the technology’s efficiency in a 3D culture, the chip recorded local concentrations and secretion rate of HGF and TGF-β1 without any disturbance for seven days (10). By potentially providing real-time monitoring compatible with adherent cells, the limited number of Ab binding sites remains a design constraint, underscoring the need for regenerable or replaceable sensing elements.
Since its invention in the 16th century, the light microscope has transformed our understanding of life, uncovering the structure of cells and the complexity of living systems. Microscopy remains one of the cornerstones of cell biology, yet it is no longer the only reliable tool for assessing cell viability and growth. Advances in analytical technology now make it possible to evaluate cell health through low-impact and high-precision methods, minimizing user bias and sampling error.
Techniques, such as pH-, lactate-monitoring, OD analysis, flow cytometry, and advanced biosensors, have demonstrated their ability to deliver reproducible quantitative data. As the CGT field expands and process budgets increase, the demand for robust bioprocess monitoring will continue to drive technological progress. None of these methods is flawless, but neither is conventional microscopy, which can be time-intensive and prone to optical limitations unless advanced imaging systems are used.
Looking ahead, microscopy will remain indispensable for discovery and detailed cellular observation. Yet, metabolic and biological sensors capable of delivering continuous high-resolution data are meant to play a far greater role in industrial-scale cell manufacturing. Together, these complementary technologies will shape a more efficient and sustainable future for cell culture monitoring.
References:
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- pH Monitoring is the Key to Cell Culture (2022). Scientific Industries, Inc. Available at https://www.evitria.com/cho-cells/hek293-cells-vs-cho-cells/ (Accessed 01 October 2025).
- pH, DO, OD, and CO₂: What each parameter measures in a cell culture (2025). TECNIC – Bioprocess Solutions. Available at: https://www.tecnic.eu/4-key-cell-culture-parameters-explained/#:~:text=pH is measured in real,ensuring a stable physiological environment (Accessed 01 October 2025).
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- Fedi, A, Vitale, C, Giannoni, P, Caluori, G, Marrella, A, Biosensors to Monitor Cell Activity in 3D Hydrogel-Based Tissue Models (2022), MDPI Sensors, 22; 4: pp. 1-34. doi: 10.3390/s22041517.
