Animal cell culture: Biology, culture environment, and characterization: A review

Introduction

Cell culture is one of the most important techniques in modern biological studies that, enables researchers to study living cells under precisely controlled in-vitro conditions. This technique involves separating cells from tissues and maintaining outside the body in a nutrient-rich culture medium that supports their survival, growth, and proliferation (Freshney, 2016) [10] . Cell culture offers a simplified and highly reproducible system that allows for more precise investigation of cellular physiology, metabolism, differentiation, and responses to biological, chemical, and physical stimuli when compared to tests conducted on whole animals. (Masters, 2000) [19] . The ability to develop cells in vitro has transformed research in the domains of microbiology, molecular biology, genetics, toxicology, pharmacology, and regenerative medicine. Under specific laboratory settings, cultured cells are useful experimental models for understanding the normal functioning of cells, assessing therapeutic efficacy and toxicity of drugs, and researching host-pathogen interactions. (Davis, 2011; Freshney, 2016) [5,10] . In line with the 3Rs (Replacement, Reduction, and Refinement) principles in biomedical research, the use of cell culture has greatly reduced reliance on experimental animals. (Russell & Burch, 1959) [23] . The invention of animal cell culture dates back to Ross Harrison's groundbreaking work in 1907, when he successfully preserved frog embryonic nerve tissue outside the body, proving that animal cells could endure and grow in the right lab settings. (Harrison, 1907) [13] . Since then, incredible advances in sterile procedures, culture media, incubator technology, and molecular biology have transformed cell culture into a vital tool in both study and industry. Cell culture is now widely employed in vaccine development, viral isolation and multiplication, monoclonal antibody production, cancer biology, stem cell research, tissue engineering, gene therapy, and a wide range of biopharmaceutical products. (Freshney, 2016;

Biology of cultured cells

Cells maintained in vitro retain many of their fundamental physiological traits, although their behavior is greatly influenced by the artificial culture environment. The numerous structural and functional alterations that take place when an organized tissue is converted into a culture vessel must be considered while designing experiments.

Reduced Cell-Cell Interactions

Cells in living tissues continuously communicate with each other through extracellular matrix proteins, gap junctions, adhesion molecules, soluble cytokines, growth hormones, and direct membrane contacts. These connections control a variety of cellular processes, including as proliferation, differentiation, migration, apoptosis, and tissue homeostasis (Alberts et al., 2022) [1] . During cell isolation, enzymatic or mechanical dissociation disrupts many of the natural cell-cell and cell-matrix interactions. As a result, cells grown in culture tend to migrate and spread more easily because they are no longer constrained by the surrounding tissue environment. In addition, the reduced contact inhibition seen in some cultured cells allows them to proliferate more actively, especially during the logarithmic (log) phase of growth. Nevertheless, prolonged culture may alter signalling pathways responsible for maintaining tissue-specific functions, resulting in changes in cellular phenotype and responsiveness to extracellular stimuli. Recent advances in physiologically relevant three-dimensional (3D) culture systems and organoid models have been developed to better preserve native cell-cell and cell-matrix interactions and overcome these limitations of conventional two-dimensional (2D) cell culture (Ko et al., 2024; Tan et al., 2024) [17,24] .

Loss of Three-Dimensional Architecture

The highly organized three-dimensional (3D) extracellular matrix (ECM) that surrounds cells in-vivo offers both mechanical support and physiological signals necessary for proper tissue function. However, the conventional twodimensional (2D) monolayer systems relies on the adherance of cells to a hard plastic surface and forms the foundation of the traditional cell culture. Absence of proper 3 D system affects the polarity of cell, its shape, cytoskeletal organization, distribution of receptors, intracellular signaling, and even gene expression. As a result, cells grown in traditional monolayer cultures often behave differently physiologically than cells found in intact tissues. (Edmondson et al., 2014) [8] . Recent advances in tissue engineering have led to the development of three-dimensional culture systems, spheroids, organoids, and organ-on-chip technologies that better reproduce the architecture and physiological characteristics of living tissues. (Fang et al., 2023) [9] .

Altered Hormonal and Nutritional Regulation

Amino acids, vitamins, glucose, inorganic salts, and buffering agents necessary for cellular metabolism are among the essential nutrients found in widely used media including Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), and RPMI-1640. These media are usually supplemented with fetal bovine serum (FBS) or chemically defined growth supplements to further support cell survival, attachment, and proliferation. These supplements provide hormones, growth factors, lipids, and other bioactive molecules that help replicate important elements of the cellular microenvironment (Freshney, 2021) [11] . Despite these supplements, cultured cells do not have the dynamic endocrine regulation present inside living organisms. Consequently, cellular metabolism, receptor expression, and differentiation patterns may differ substantially from their physiological counterparts.

Changes in Cellular Differentiation

Specialized cells inside tissues perform certain physiological functions that are maintained by continuous interactions with extracellular matrix components and nearby cells. After being isolated and repeatedly subcultured, many initial cells gradually lose their differentiated phenotype; this process is known as dedifferentiation. For example, primary hepatocytes eventually lose their ability to generate albumin and metabolize xenobiotics, while chondrocytes may cease producing extracellular matrix proteins specific to cartilage after prolonged monolayer culture. When evaluating experiments, these changes must be considered because they reduce the physiological importance of grown cells (Lodish et al., 2021) [18] . Modern culture techniques employing extracellular matrix coatings, co-culture systems, and threedimensional scaffolds have significantly improved the maintenance of differentiated cellular functions (Duval et al., 2017) [17] .

Limited Lifespan of Primary Cells

Most primary animal cells have a limited proliferative capacity. They go into a state of irreversible growth stoppage called replicative senescence after a certain number of cell divisions. This phenomenon, first described by Hayflick and Moorhead (1961) [14] , is primarily attributed to progressive telomere shortening and accumulation of cellular stress. Senescent cells continue to be metabolically active but stop proliferating.

They frequently exhibit distinctive morphological changes, such as increased granularity, larger cell size, and changed gene expression. As a result, primary cultures are best suited for brief studies that need to be physiologically relevant. For survival and proliferation, several cell types-such as fibroblasts, endothelial cells, and epithelial cells-need to be attached to a solid substrate. On treated plastic surfaces, these cells typically develop as monolayers.

Suspension Cells

Many hematopoietic and lymphoid cells are inherently adapted to grow in suspension, in contrast to anchoragedependent cells. Because these cells either dwell in situations where attachment is not necessary or circulate freely in the circulation in-vivo conditions, therefore, a surface that promotes cell attachment is not necessary for the successful growth of these cells in suspension. Suspension cultures provide a number of useful benefits, including as more consistent exposure to nutrients and dissolved gasses, simpler scale-up for bioreactor production, and simpler harvesting. For vaccine development, antibody manufacturing, and immunological research, a variety of hybridoma, leukemia, lymphoma, and immune cell lines are frequently cultured as suspension cultures. (Davis, 2011) [5] .

The Cell Culture Environment

For cultured cells to be successfully maintained, physiological conditions must be precisely replicated. Numerous environmental factors impact development, survival, and functional integrity of the cells maintained in cell culture.

Temperature

The optimal incubation temperature generally reflects the normal physiological temperature of the organism from which the cells originate. Mammalian cells are routinely cultured at 37°C, corresponding to their average body temperature (Freshney, 2016) [10] . Avian cells require slightly higher temperatures, typically 39-41 °C, to support optimal growth and cellular function (Pattison et al., 2008) [21] . In contrast, insect cell lines are commonly maintained at 27-28 °C, which closely matches their natural physiological conditions and supports efficient growth and recombinant protein production (Merten, 2015) [20] . Deviations from the optimum temperature adversely affect membrane fluidity, enzyme activity, protein synthesis, and cellular metabolism (Freshney, 2016) [10] . pH Most mammalian cells grow optimally at a pH between 7.2 and 7.4. The pH is commonly maintained using bicarbonatecarbon dioxide buffering systems or HEPES buffer. Insect cell cultures are generally maintained at a slightly lower pH of 6.2-6.4, reflecting the conditions of insect hemolymph. Unlike mammalian cells, most insect culture media (e.g., Sf-900 or Grace's Insect Medium) are buffered with phosphate and organic buffers instead of bicarbonate. Even slight fluctuations in pH influence enzyme activity, nutrient transport, and cell viability (Freshney, 2016) [10] .

Carbon Dioxide

Culture media containing sodium bicarbonate require incubation in an atmosphere containing approximately 5% CO₂ to maintain physiological pH especially for Mammalian and avian cell cultures. In contrast, most insect cell cultures are maintained without supplemental CO₂ because their media are buffered with phosphate or organic buffering systems that remain stable under atmospheric conditions. (Thermo Fisher Scientific, 2021) [25] .

Oxygen

Cellular respiration, energy production, and proliferation are all influenced by oxygen availability. While most cell cultures are kept at atmospheric oxygen levels (roughly 18-21% O₂), many primary cells and stem cells show better growth and physiological function under lower oxygen conditions (2-10% O₂), which more closely resemble the in vivo tissue environment. As a result, oxygen requirements may differ depending on the type of cell and the goal of the experiment (Freshney, 2016) [10] .

Humidity

In order to reduce evaporation of the culture media and preserve consistent osmolarity, high humidity of approximately 95% is maintained in cell culture incubators (Freshney, 2016) [10] .

Sterility

One of the most important prerequisites for animal cell culture is the maintenance of aseptic conditions. Bacterial, fungal, yeast, mycoplasma, or viral contamination can change cellular metabolism, affect the expression, and eventually ruin the entire experiment. As a result, all processes involving cultivated cells must be carried out in biological safety cabinets, utilizing sterile media, chemicals, plasticware, and equipment. (Uphoff & Drexler, 2014) [27] . Amphotericin B (0.25-2.5 μg/mL) may be added when fungal contamination is a concern, although antibiotics like penicillin (100 U/mL) and streptomycin (100 μg/mL) are frequently added to the culture media of many mammalian and avian cell lines to lower the risk of bacterial contamination. Although antibioticantimycotic combinations may be used during primary culture establishment or when contamination is suspected, insect cell cultures are often maintained without antibiotics under proper aseptic conditions. (Freshney, 2016) [10] .

Characterization of Cultured Cells

Accurately characterizing growing cells is crucial in cell culture studies. Reliable identification ensures the biological importance, credibility, and repeatability of experimental outcomes. Over the years, a number of studies have reported cell line misidentification and cross-contamination, leading to inaccurate scientific conclusions and a substantial loss of research resources. (Capes-Davis et al., 2010; ATCC Standards Development Organization Workgroup ASN-0002, 2010) [4,3] . Consequently, international organizations such as the American Type Culture Collection and the International Cell Line Authentication Committee recommend routine authentication of cell lines before their use in research (Freshney, 2016; Almeida et al., 2016) [10,2] . Characterization involves determining the identity and biological properties of cultured cells, including their: • morphology, • species of origin, • tissue source, • differentiation status, • genetic stability, and • expression of lineage-specific markers.

No single method is sufficient for complete identification; instead, a combination of morphological, cytogenetic, biochemical, immunological, and molecular techniques provides the highest degree of accuracy.

Morphological Characterization of Cultured Cells

Examining the morphology of grown cells with an inverted phase-contrast microscope is the first step in identifying them.

Cell morphology provides important initial information about a culture's identification, physiological state, and growth traits. However, morphological observations should always be interpreted cautiously because a number of factors, including cell density, culture medium composition, substrate properties, passage number, and ambient conditions, affect cell shape. (Freshney, 2016) [10] . Cells maintained under optimal culture conditions generally display characteristic shapes that permit tentative classification into three major morphological categories.

Morphological category Morphological characteristics Growth characteristics Typical tissue origin Representative cell lines

Species Identification of Cultured Cells

To ensure experimental reproducibility and avoid crossspecies contamination, it is crucial to identify the species of origin. Chromosome and isoenzyme analysis were the methods used previously for species identification. Today, molecular genetic techniques have largely replaced these classical approaches because of their superior sensitivity and specificity.

Common approaches include Chromosome Analysis

Cytogenetic differentiation is made possible by the diverse chromosome counts and karyotypes of various animal species.

Chromosomal anomalies or alterations due to cellular transformations or prolonged culture in vitro can be detected by Karyotyping and offer important information on genomic stability. However, this method requires actively dividing cells, competent personnel, and superior metaphase preparations that limits use of this method (Freshney, 2016) [10] .

Isoenzyme Analysis

The electrophoretic mobility of isoenzymes varies depending on the species. It is possible to distinguish between closely related species by analyzing enzymes including lactate dehydrogenase (LDH), malate dehydrogenase (MDH), glucose-6-phosphate dehydrogenase (G6PD), and peptidase B.

DNA-Based Identification

For species authentication, molecular genetic methods are currently the gold standard. For precise identification of cultured cells, modern labs are increasingly using PCR amplification, DNA sequencing, mitochondrial DNA analysis, and species-specific molecular probes. These techniques provide rapid and highly reliable results while requiring only small quantities of DNA (Almeida et al., 2016) [2] .

Identification of Tissue of Origin

Determining the tissue or organ from which the cells originated is the next goal after proving species identity. In research involving stem cells, cancer biology, regenerative medicine, and developmental biology, understanding tissue genesis is very crucial. The following are the main standards for tissue identification:

• Cellular growth features and morphology

Cell adhesion and extracellular matrix interaction

Monoclonal antibodies directed against these antigens are widely used in flow cytometry and immunocytochemistry for cell identification.

Characterization of Transformed Cells

The acquisition of genetic changes that lead to permanent shifts in cellular phenotype is referred to as transformation. Because transformed cells differ substantially from normal primary cells, their identification is essential when selecting experimental models.

Importance of Cell Line Authentication

Since continuous and long term passaging may cause certain phenotypic and genetic changes in the original cell line, authentication is very crucial. Routine authentication offers several advantages:

• Ensures reproducibility of experimental results.

• Detects interspecies and intraspecies crosscontamination.

• Confirms the genetic identity of cultured cells.

• Prevents publication of misleading scientific data.

Detection of Microbial Contamination

Microbial contamination is one of the leading causes of experimental variability in cell culture laboratories. Common contaminants include-Bacteria, Virus, Fungi, Yeasts and Mycoplasmas. Among these, mycoplasmas are particularly problematic because they do not produce visible turbidity and frequently remain undetected while altering cellular metabolism, DNA replication, gene expression, etc. Routine screening methods include-PCR, DNA fluorochrome staining (Hoechst or DAPI), culture techniques and enzymatic detection assays. Regular monitoring combined with strict aseptic practices significantly reduces contamination risks.

Cell Adhesion and Extracellular Matrix Interactions in Cell Culture

Cell adhesion is a fundamental processes in biology that enables cells to attach to their neighboring cells and to the extracellular matrix (ECM). These interactions are not just a passive attachment, its deeply related to how tissues maintain their shape and how cells carry out regular cellular functions such as cell division, migration, differentiation into specialized types, survival, and intracellular signaling. In cell culture, successful adhesion is particularly important because cells that fail to adhere often struggle to maintain their normal physiological characteristics and eventually die. Consequently, understanding the mechanisms by which cells adhere is crucial for applications in modern research involving cell biology, regenerative medicine, tissue engineering, cancer research, and vaccine production (Hynes, 2002; Freshney, 2016) [15,10] . Most cells isolated from solid tissues-fibroblasts, epithelial cells, endothelial cells, and mesenchymal stem cells-usually donot grow unless they attach to some solid surface, hence are called anchorage-dependent. After seeding, these cells start establishing a weak contact with the culture vessel which in due course of time convert into a firm attachment through interactions between cell surface receptors and extracellular matrix proteins. Once attached, cells then reorganize their cytoskeleton to spread out across the surface eventually enter the cell cycle. Cells originating from blood, bone marrow, spleen, and lymphoid tissues behave differently. They generally grow in suspension because no attachment is needed for their survival or proliferation (Masters, 2000) [19] . When anchorage-dependent cells fail to establish proper adhesion, they often undergo a specialized form of programmed cell death known as anoikis and plays an important role in maintaining normal tissue organization. However, many transformed and metastatic cancer cells manage to develop resistance to anoikis, allowing them to survive during circulation and invade distant tissues (Frisch & Francis, 1994) [12] . The extracellular matrix (ECM) is a highly organized scaffold of proteins and polysaccharides that surrounds cells in living tissues. Besides providing structural support, it plays an important role in regulating cell behaviour-migration, differentiation, proliferation, and survival. The major components of the ECM include collagen, laminin, fibronectin, elastin, proteoglycans, and glycosaminoglycans. In conventional two-dimensional cultures, the native ECM is largely absent, which explains why cultured cells often behave differently from cells in vivo. To overcome this limitation, culture surfaces are frequently coated with ECM proteins such as collagen, fibronectin, laminin, or gelatin to improve cell attachment and maintain specialized cellular functions (Hynes, 2009) [16] .

Cell Adhesion Molecules (CAMs)

Cell adhesion is mediated by specialized transmembrane proteins collectively known as cell adhesion molecules (CAMs). These molecules in addition to providing mechanical attachment, also activate signaling pathways inside the cells that regulate various cellular activities.

Integrins

Among the different families of CAMs, integrins are the principal receptors responsible for cell-ECM interactions. They bind to extracellular matrix proteins such as collagen, fibronectin, laminin, and vitronectin through specific recognition motifs. Binding of integrins activates signaling pathways thereby regulating cell proliferation, migration, differentiation, and survival (Ruoslahti, 1996; Hynes, 2002) [22, 15] .

Cadherins

Cadherins constitute another important family of adhesion molecules that primarily mediate calcium-dependent cell-tocell adhesion. They hold tissue at structural level, forming adherens junctions between adjacent cells (Thiery et al., 2009) [26] . Other adhesion molecules also contribute to cellular interactions-Selectins, members of the immunoglobulin superfamily and proteoglycans, etc. Recent advances in cell culture has centered on one goalrecreating the native extracellular environment. Threedimensional (3D) culture systems, biomaterial scaffolds, organoids, and organ-on-chip technologies mimic the architecture and mechanical properties of living tissues much better than the conventional monolayer cultures by better preserving cell-cell and cell-matrix interactions (Edmondson et al., 2014) [8] .

Conclusion

The successful maintenance of cultured cells depends on setting up conditions that mirror their natural physiological environment. Cells require an appropriate culture medium containing essential nutrients, proper physical and chemical conditions to ensure normal cellular growth and function (Masters, 2000) [19] . Strict aseptic techniques are equally important because contamination by bacteria, fungi, mycoplasma, or viruses can compromise experimental results and lead to loss of valuable cell cultures. A clear understanding of extracellular matrix interactions and adhesion molecules is also essential for maintaining healthy cell cultures and for advancing research in developmental biology, tissue engineering, oncology, immunology, and

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