INTRODUCTION

Cell biology, also known as cytology, is the scientific discipline dedicated to understanding cellular structure, function, physiology, and biochemical behavior. A core methodological approach within this field is cell culture, which involves the maintenance and propagation of cells in vitro following their transfer from an original biological source or an established culture. Cell culture systems form the experimental backbone of modern biomedical research and are extensively applied in vaccine production, drug and compound screening, toxicology testing, molecular biology, and regenerative medicine research [1]. Their primary advantage lies in the ability to precisely control the extracellular environment, thereby enabling reproducible investigation of cellular responses under defined conditions.

The successful propagation of mammalian cells in vitro depends on the optimization of both physiological parameters and growth medium composition. Key physiological factors include temperature, atmospheric gas composition, and humidity. Mammalian cells are typically maintained at approximately 37 °C to replicate physiological body temperature, while controlled humidity minimizes evaporative loss from culture vessels. A carbon dioxide (CO₂) concentration of around 5% is routinely employed to stabilize extracellular pH and support optimal cellular metabolism [2]. Together, these parameters recreate essential aspects of the in vivo cellular microenvironment and are critical for sustaining cell viability, proliferation, and functional stability.

Growth media supply the biochemical components required for cellular survival and division. Essential constituents include carbohydrates such as glucose, amino acids, vitamins, inorganic salts, and buffering agents, most commonly bicarbonate. Glucose and glutamine serve as major energy substrates, while amino acids function as building blocks for protein synthesis. Vitamins support enzymatic activity and redox balance, and ionic solutions maintain osmotic pressure and membrane potential within the culture system. The composition of the growth medium, in conjunction with controlled temperature and gas exchange, governs nutrient availability, waste removal, and intracellular signaling pathways [3].

Carbon dioxide is a colorless and odorless gas with a molecular weight of 44.01 g/mol, composed of a single carbon atom covalently bonded to two oxygen atoms. Although atmospheric CO₂ represents only approximately 0.04% of the Earth’s atmosphere, it plays a fundamental role in cellular metabolism and physiological regulation. In mammalian cell culture, CO₂ is primarily used to regulate pH through equilibrium with bicarbonate-based buffering systems, closely mimicking physiological extracellular conditions [4]. This buffering mechanism is essential for maintaining enzyme activity, membrane stability, and metabolic homeostasis.

In addition to pH regulation, CO₂ is intrinsically linked to aerobic metabolism and cellular respiration in both microbial and mammalian systems. It is an inevitable end product of oxidative metabolism and accumulates during cellular growth and bioprocessing. In large-scale cell culture and bio manufacturing platforms, dissolved CO₂ concentrations are influenced by cellular metabolic activity, physical constraints of the culture system, hydrostatic pressure, aeration efficiency, and bioreactor design [5]. Elevated CO₂ levels can alter intracellular pH, protein conformation, metabolic flux, and transcriptional regulation, thereby impacting cell growth and productivity.

Emerging evidence further suggests that CO₂ may function beyond a passive metabolic by-product, acting as a regulatory signal that modulates gene expression and cellular stress responses. CO₂-mediated effects on transcriptional networks and metabolic pathways have been reported in both microbial and mammalian cells, highlighting its broader biological relevance [6]. Collectively, the physicochemical properties of CO₂ exert substantial influence on cellular physiology, metabolic regulation, and bioprocess performance (Figure 1). A comprehensive understanding of these interactions is essential for optimizing cell culture systems and improving outcomes in biomedical research and therapeutic production. An overview of the role of CO₂ in optimizing cell culture conditions [7-11].

Importance of CO₂ in Cell Culture

Carbon dioxide (CO₂) is a critical component of modern cell culture systems and plays an indispensable role in maintaining optimal in vitro growth conditions. Contemporary CO₂ incubators are designed to regulate multiple environmental parameters simultaneously, including temperature, humidity, and gaseous composition, thereby closely mimicking physiological conditions. The primary functions of CO₂ in cell and tissue culture include maintenance of a stable physiological pH, preservation of a humid and sterile environment, and indirect support of optimal cellular metabolism. Depending on the specific application and cell type, CO₂ concentrations are typically maintained within a range of 3%–10%, with 5% being the most widely adopted standard for mammalian cell culture systems [12].

The principal role of CO₂ in cell culture is to sustain a steady-state physiological pH through a bicarbonate-based buffering system. CO₂ from the incubator atmosphere readily dissolves in the aqueous culture medium, where a fraction reacts with water to form carbonic acid. This reaction establishes an equilibrium between dissolved CO₂, carbonic acid, bicarbonate ions, and hydrogen ions, thereby regulating extracellular pH. Most mammalian cell lines exhibit optimal growth within a narrow pH range of 7.2–7.4, which closely resembles physiological conditions in vivo. Deviations from this range can adversely affect enzyme activity, membrane integrity, and overall cellular viability [3]. The fundamental components of a standard cell culture medium and their functions are summarized in Table 1.

CO₂ Levels and pH Regulation in Cell Culture

The pH of cell culture systems is primarily governed by the buffering capacity of the growth medium, which protects cells against abrupt fluctuations in acidity or alkalinity. Both organic buffers and CO₂–bicarbonate buffering systems are employed; however, the latter remains the most physiologically relevant and widely used approach. Because pH regulation depends on a delicate balance between dissolved CO₂ and bicarbonate ions (HCO₃⁻), alterations in atmospheric CO₂ concentration can directly influence medium pH. Consequently, precise control of incubator CO₂ levels is essential for experimental reproducibility and cell health [13].

Most routine mammalian cell culture experiments utilize atmospheric CO₂ concentrations ranging from 5% to 7%, although some specialized applications require levels as low as 4% or as high as 10%. The sodium bicarbonate concentration in the culture medium determines the exact CO₂ level needed to maintain physiological pH. Media containing higher bicarbonate concentrations require correspondingly higher CO₂ levels to achieve equilibrium. This interdependence underscores the importance of matching medium formulation with incubator settings to ensure stable culture conditions [3]. The roles of CO₂ in biomedical and medical applications related to cell culture are outlined in (Table 2) [14,15].

CO₂ Monitoring and Medical Applications

Carbon dioxide is ubiquitous in both environmental and biological systems, and its accurate measurement has long been a focus across diverse scientific disciplines. CO₂ sensing technologies are employed in numerous applications, including food preservation, agricultural systems, environmental monitoring, analytical chemistry, and biomedical research. In medical science, CO₂ monitoring is particularly critical, as it provides essential information regarding respiratory and circulatory function. Monitoring CO₂ levels in patients offers valuable insight into ventilation efficiency, metabolic status, and overall physiological stability [16].

Clinically, CO₂ monitoring is performed using three primary approaches: arterial blood gas analysis, airway capnometry, and transcutaneous capnography. Among these, non-invasive techniques—particularly transcutaneous and fluorescence-based CO₂ sensing—have gained increasing attention due to their potential for continuous, real-time monitoring without patient discomfort. Accurate assessment of CO₂ levels, in conjunction with oxygen measurements, enables clinicians to evaluate respiratory adequacy and detect early signs of physiological compromise [17].

In critical care, emergency medicine, and anesthesiology, CO₂ measurement is a well-established standard for monitoring respiratory function. Advanced multi-sensor platforms now integrate CO₂ sensing with additional physiological parameters, allowing comprehensive, multiparametric patient monitoring in intensive care settings. These technologies enhance clinical decision-making and contribute to improved patient outcomes by providing continuous, high-resolution data on vital physiological processes [18].

DISCUSSION

Carbon dioxide (CO₂) occupies a central position in contemporary biomedical research due to its indispensable role in maintaining tightly controlled cell culture environments. Precise regulation of CO₂ concentration is fundamental for preserving sterility, physiological pH, and optimal metabolic conditions, all of which are prerequisites for reliable in vitro experimentation. In mammalian cell culture systems, deviations in CO₂ levels can result in pH instability, impaired cellular metabolism, and compromised experimental reproducibility, underscoring the importance of continuous monitoring and environmental control [19].

The relevance of CO₂-regulated culture systems is particularly evident in stem cell research, regenerative medicine, and translational clinical applications. CO₂ incubators provide a controlled microenvironment that supports the expansion and differentiation of human cells for therapeutic use. These systems enable the cultivation of autologous and allogeneic cells under conditions that closely resemble the in vivo physiological milieu, thereby preserving cellular functionality and genetic stability. Applications include the expansion of human keratinocytes for skin grafting, generation of stem cells for disease modeling, and development of tissue constructs for reconstructive and plastic surgery [20,21].

Regenerative medicine represents one of the most promising frontiers in modern healthcare, with CO₂-regulated cell culture serving as a foundational technology. By enabling the controlled proliferation of patient-derived cells, regenerative approaches facilitate the repair or replacement of damaged tissues and organs. In the context of severe burn injuries, autologous skin cell expansion in CO₂ incubators allows for transplantation with reduced immunogenicity and minimal scarring compared with conventional grafting techniques. This strategy exemplifies how optimized CO₂ environments directly contribute to improved clinical outcomes [22].

Advances in stem cell biology further highlight the importance of CO₂ control in disease treatment, particularly in oncology and cardiovascular medicine. In vitro–generated tissues and stem cell–derived constructs are increasingly explored for repairing cardiac tissue, reducing the need for invasive surgical interventions. In such applications, cells are cultured, expanded, and functionally validated under CO₂-regulated conditions before transplantation. Both autologous approaches—using patient-derived cells—and bioengineered tissues fabricated de novo rely on stringent CO₂ control to ensure safety, viability, and therapeutic efficacy [23].

Maintaining sterility throughout cell culture workflows remains a critical concern, especially during cell isolation, expansion, and genetic testing stages. CO₂ incubators equipped with automated sterilization systems significantly reduce the risk of microbial contamination, thereby enhancing biosafety and compliance with clinical manufacturing standards. This is particularly important in personalized cell therapies, where cells undergo multiple processing steps prior to implantation. Comprehensive sterility and genetic stability assessments conducted within controlled CO₂ environments are essential to minimize clinical risks and ensure therapeutic reliability [24].

Beyond regenerative medicine, CO₂-regulated systems support a wide array of biomedical applications, including dermatological treatments, orthopedic tissue repair, and anti-aging therapies. The capacity to expand patient-specific cells under physiologically relevant conditions enables personalized treatment strategies while reducing immune rejection. Collectively, these applications demonstrate that CO₂ incubators are not merely supportive tools but essential platforms driving innovation across multiple domains of modern medicine [25].

CONCLUSION

Cell culture technologies are integral to modern biomedical research, providing critical insight into cellular behavior, disease mechanisms, and therapeutic development. Among the environmental parameters governing cell culture systems, carbon dioxide plays a particularly significant role due to its direct influence on intracellular pH regulation, metabolic activity, growth kinetics, and product quality. CO₂ functions not only as a buffering agent but also as a regulatory signal that affects gene expression and cellular homeostasis in both microbial and mammalian systems. Appropriate control of CO₂ concentration and pH is therefore essential for maintaining experimental consistency, optimizing cellular productivity, and ensuring translational relevance. As cell-based therapies and regenerative medicine continue to evolve, the importance of optimized CO₂ management will further increase. Careful integration of CO₂ regulation strategies into culture system design will facilitate advancements in tissue engineering, stem cell therapy, and precision medicine. Overall, maintaining optimal CO₂ levels is fundamental to enhancing cellular metabolism, process performance, product quality, and the long-term success of cell culture–based biomedical applications.

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