Inferences of Carbon Dioxide in Present-Day Cell Culture Systems: An Unacknowledged Problem and Perspectives

Review Article

Austin Therapeutics. 2021; 6(1): 1033.

Inferences of Carbon Dioxide in Present-Day Cell Culture Systems: An Unacknowledged Problem and Perspectives

Dubey AK1, Lavanya L2, Sadananda D3, Gouthami K4, Elfansu K5, Singh A6, Singh A7 and Singh AK8*

1Department of Biotechnology, Indian Institute of Technology Madras, India

2Department of Biochemistry, REVA University, India

3WPU Division, Institute of Wood Science and Technology, India

4School of Applied Sciences, REVA University, India

5Division of Molecular Biology, JSS Academy of Higher Education and Research, India

6Medicinal and Process Chemistry, CSIR-Central Drug Research Institute, India

7ISF College of Pharmacy, India

8Department of Pharmacology, Pennsylvania State University, Penn State College of Medicine, USA

*Corresponding author: Ashok K Singh, Department of Pharmacology, Post-doctoral Research Trainee, Pennsylvania State University, Penn State College of Medicine, Hershey, PA, USA

Received: February 17, 2021; Accepted: March 13, 2021; Published: March 20, 2021


The impact of cell culture technology has been immense in the field of research and pharmaceutical sciences. Suitable environmental conditions are paramount need in order to maintain the cell line in an in-vitro condition. Since the biological processes are highly susceptible to acid-base chemistry, variations among the factors will have a severe effect on cellular integrity that often leads to hypercapnia and senescence of the cells. pH regulation in the cell culture environment is a fundamental biological phenomenon of great significance for the growth and metabolism of cells. This review focuses on the implications of the two critical factors carbon dioxide (CO2), pH, and their correlated effects in the mammalian culture system. Bicarbonate buffer plays a vital role in maintaining homeostasis as dissolved CO2 hydration occurs in bicarbonate (HCO3-) and H+ equilibriums. If pH is not controlled, inhibition of CO2 causes acidification in the medium. In contrast, if pH is not regulated by integrating essential requirements, the equilibrium reaction shifts to the right towards the hydrogen ions that maintains the balance. Despite a few literatures exhibiting the role of carbon dioxide in cell culture, the present review distinguishes from them by showing the effects of CO2 in the in-vitro environment in maintaining the pH balance and cellular integrity. Thus, ensuring the proper mechanism of the utilization of CO2 and pH in the cell culture system will undoubtedly lead to the exploration of enormous concepts in the present crucial study.

Keywords: Hypercapnia; Senescence; Cellular metabolism; Bicarbonatebuffering system; Cell density; Intelligent fail-safe system


The condition of growth is major characteristics of living organisms and impersonating this technology in-vitro could be possible through tissue culture, which requires appropriate environmental conditions and culture medium in either liquid or solid form. Growth of tissue or cell separation of an organism is capable of evidently growing in invitro conditions from living tissues. Various researchers are trying to give presentable work in the field of animal cell cultures [1]. Primary cells proliferating from organs or tissue in the in-vitro environment resemble the parental cells, which reflect changes in the growing cells, organs, or tissues. Cell proliferation requires a suitable environment and type of media for culturing, and with the right constituents, the process becomes more accessible to culture, which is meant to achieve [2].

Suitable environmental conditions are significantly responsible for cell maintenance and growth. Cell culture technique has a considerable advantage to manipulate and reproduce under optimum environmental conditions [3]. In a simulated environment, the cell culture system involves mainly factors such as pH, ideal gas, temperature, and humidity, which allows cells to reproduce and grow. However, primary cells are typically preferred over continuous cell lines due to their physiological similarity in correspondence to the cells from living tissue. Furthermore, continuous cell lines undergo specific phenotypic and genotypic changes that lead to discrepancies during analysis. The cells obtained from living tissue in resemblance of primary cells, necessary for research can be studied for their functions, development, defects, metabolic regulations, cell physiology, and conditions affecting the tissue of interest [4].

While working with human tissue, which is frequent pathogenic, biosafety, is essential, especially with the range of equipment required in carrying out the isolation of cells; purification and culture, as described for tissue culture laboratory. However, precautions are required to avoid infection of species by microorganisms in order to preserve aseptic conditions. Understanding basic cell-culture handling techniques, including suitable choice of culture media, and cryopreservation can be an essential factor in maintaining cultures. Thus, culture techniques can be significantly employed for screening drugs, disease diagnosis, and the development of vaccines [5]. Despite the plethora of research articles published in correlating the CO2 and the cell culture system, there is no review so far present in the literature that describes the critical phenomenon of the effect of CO2 in the in-vitro environment in maintaining the cellular integrity of the cells in our knowledge. The idea of this study was considered when we observed in the growing fibroblast cell lines under experimental conditions with the variation of CO2 levels inside the incubator. As the level of CO2 dropped below the optimum value of 5%, there were physiological changes observed in the morphology of the cell lines that finally lead to the loss of the adherence property. The color of the medium was also observed for the changes with variation in the CO2 levels, which could be due to the variation in the pH, which is intercorrelated. This review emphasizes the correlative study of these two factors inside the cell culture system and their effects in-vitro.

Cell Culture Media

Cell culture media has significant advantages in the manipulation of the physiological environment (hormone and nutrient concentration) and physicochemical conditions (pH, temperature, O2, osmotic pressure, and CO2 tension) for cell proliferation. For both growth and maintenance, proper cell culture environment is a necessity [1]. For the cells to grow in-vitro and maintain their life, it is essential to get it supplemented with all types of nutrient media. The balanced salt solution contains a proper mixture of nutrients to regulate optimum osmotic pressure and provides essential nutrition to the cells. The components of the cell culture medium include inorganic salts, serum, glucose, vitamins, and amino acids. The serum is used as growth factors, hormones, and attachment factors [6]. In the enzymatic reactions, metal ions act as co-factors and improve cell adhesion. Phenol red used as pH indicator, at the range pH 7.0-7.4 changes from orange (red) to yellow in acidic conditions and turn to purple in basic condition. The buffer solution used for pH balancing products, e.g., the Bicarbonate/HEPES buffer for normal growth conditions, retains precise parameters such as temperature (37°C), CO2 supplement (5%) and relative humidity (95%) [7]. Transformed cell lines can see better growth at the slightly acidic condition at pH 7.0-7.4 and, whereas the basic state, substantiates a pH range of 7.4- 7.7. Carbon dioxide regulates the pH in the medium, and balances bicarbonate (HCO3-) and changes in the atmospheric CO2. A concentration of 5% of CO2 is usually maintained in the air, and 10% of CO2 is mostly used for experimental purposes [8].

Carbon functions as a source of energy, such as glutamine/ glucose. Organic compounds such as amino acids contain carboxyl (-COOH) and amine (-NH2) as a functional group along with the side chain [9]. Each amino acid contains R-group as specific [10]. The amino acid is the main component of the protein-synthesizing process. To promote the building blocks of proteins, it must contain vital elements of amino acid such as N2, C, and O2. For many enzymes, vitamins act as co-factors, and its function is essential in advancing cell growth and survival. Cell death or loss of productivity occurs due to absorbed vitamins. e.g., Vitamin-C, Vitamin-B2, Vitamin-B6, Vitamin-B7, etc. Basal media contains serum, as a source of growth and adhesion factors regulating the permeability of cell membranes and acts as a carrier of proteins, enzymes, lipids, micronutrients, and some trace elements [11] (Table 1).