Essential Aspects of Animal Cell Culture
Animal Cell Culture Essentials
A well-equipped laboratory is fundamental for successful animal cell culture. Here is a list of essential equipment:
Basic Equipment for Animal Cell Culture
- Sterile Work Area/Cell Culture Hood (e.g., laminar-flow hood or biosafety cabinet)
- Incubator (humid CO2 incubator recommended)
- Water Bath
- Centrifuge
- Refrigerator and Freezer (–20°C)
- Cell Counter (e.g., automated cell counter or hemocytometer)
- Inverted Microscope
- Liquid Nitrogen (N2) Freezer or Cryostorage Container
- Sterilizer (e.g., autoclave)
Sterile Work Area Requirements
To maintain aseptic cell culture conditions, it is crucial to prepare a separate room or dedicated space. This area should be free from traffic and, if possible, equipped with an airflow cabinet that provides filtered air to the work surface. While a HEPA (High-Efficiency Particulate Air) filtered air system is ideal, it may not always be economical. The laboratory must be specifically designated for clean culture work, and the culture of primary animal tissue and microorganisms should be strictly restricted in or near the cell culture laboratory.
Understanding Cell Lines
Continuous cell lines have acquired the ability to proliferate indefinitely through random mutation, as seen in transformed cancer cell lines, or deliberate modification, such as artificial expression of cancer genes (immortalized). Primary cells are more delicate and challenging to work with than continuous cell lines. Continuous cell lines offer limitless growth potential and are a quick and easy way to obtain basic information. However, working with continuous cell lines has some disadvantages, including their genetically modified/transformed nature, which can alter physiological properties and render them unrepresentative of the in vivo state. These characteristics can also change over time with extensive passaging.
Defining Cell Lines
A cell line is a group of cells derived from a pure cell culture subculture in primary culture. Cell lines typically have functional properties similar to primary cells, but their genotype and phenotype can differ. A cell line is a collection of cell lineages with similar or dissimilar phenotypes. Essentially, a cell line is a long-established cell culture that will continue to proliferate indefinitely if given adequate fresh medium and space. Lines, as opposed to cell strains, become immortalized. Cell culture and cell lines have grown significantly in importance for studying physiological, pathophysiological, and differentiation processes in specific cells.
Monolayer vs. Suspension Culture
Tissue culture is a methodology used to induce the growth of cells separated from an organism and grown in various culture media. In tissue culture, the culture media play a vital role. Different cells thrive in different culture media depending on their physiological and chemical features. Monolayer culture and suspension culture are two main types of cultures used in tissue culture. A monolayer culture is an anchorage-dependent culture where cells grow while attached to a substrate, whereas suspension culture is an anchorage-independent culture where cell aggregates are used to establish cell cultures in liquid media. This is the key difference between monolayer culture and suspension culture.
What is Monolayer Culture?
Monolayer culture is defined as a type of culture in which cells are cultivated in a single layer on a flask or petri dish containing the culture medium. Monolayer culture is also referred to as adherent culture or anchorage-dependent culture. The reason for referring to it as adherent or anchorage-dependent culture is that these cells require a substrate for attachment during their growth; they adhere to the cell culture vessel containing the substrate.
The substrates utilized in the culture medium should be charged prior to usage. These charged substrates promote cell-to-cell interactions. Electric ion dischargers, divalent cation coatings, and gamma irradiation are sources used to charge these substrates. In monolayer culture medium, once the cells are inoculated, their growth is regulated by a process called contact inhibition. During contact inhibition, the proliferation of normal cells is arrested once they have formed a monolayer attached to the surface.
What is Suspension Culture?
A suspension culture can be defined as a type of culture where the multiplication of small aggregates of cells or single cells takes place, suspended in a liquid medium that is constantly agitated. In other terms, suspension culture is also known as cell culture of cell suspension culture. This culture assists in the establishment of single-cell cultures that can be utilized during the investigation of plant cells with respect to their different potentials and properties. Investigations into this aspect can be used to understand the interrelationships between cells and their influences within multicellular organisms.
During initial plant development, a callus forms as a mass of undifferentiated cells. This growth level causes obstacles for the identification of cellular events during the growth and development phases of the plant. Suspension culture was introduced to overcome this situation, as it provides an excellent culture medium for the establishment of single-cell cultures from small cell aggregates obtained from the callus. This technique has been utilized to study different morphological and biochemical aspects during the initial stages of a plant’s development.
In the context of obtaining an ideal cell culture, the typical method is to transfer a friable mass of callus into an agitated liquid medium where the mass is broken and dispersed readily. Afterwards, the large calli are screened out, keeping the small cell aggregates and single cells. Then these cells are transferred into another medium. Actively growing cells are typically obtained after two to three weeks.
Characterization of Cultured Cells
Characterization of cultured cells is a crucial step in cell biology and biomedical research. This process involves a comprehensive analysis of cell properties to ensure their identity, purity, and functionality. Without proper characterization, research findings can be compromised, leading to inaccurate conclusions and wasted resources. Therefore, investing time and effort into cell characterization is essential for reliable and reproducible results.
Identity Verification
One significant aspect of cell characterization is verifying the cell line’s identity. Over time, cell lines can become contaminated or misidentified, leading to the use of incorrect cell types in experiments. Techniques such as short tandem repeat (STR) profiling are commonly used to confirm cell identity. Consequently, researchers can be confident in the accuracy of their experimental models, which is fundamental for the validity of their findings.
Purity Assessment
In addition to identity verification, assessing the purity of cultured cells is vital. Contamination with other cell types or microorganisms can significantly impact experimental outcomes. Regular testing for mycoplasma and other contaminants helps maintain the integrity of cell cultures. Furthermore, monitoring for genetic stability is important, as prolonged cell culture can lead to genetic drift. This ensures that the cells used in experiments retain their original characteristics over time.
Functional Characterization
Moreover, functional characterization provides insights into the biological behavior of cultured cells. Researchers evaluate various cellular functions, such as proliferation, differentiation, and response to stimuli. These assessments are crucial for understanding the physiological relevance of the cell model to human biology. For instance, in drug development, knowing how cultured cells respond to potential therapeutic compounds is critical for predicting clinical efficacy and safety.
Morphological Analysis
Another key aspect of cell characterization is the analysis of cell morphology. Morphological assessment involves examining cell shape, size, and structure under a microscope. Changes in cell morphology can indicate underlying alterations in cell health or behavior. Thus, regular morphological checks are essential for maintaining healthy cell cultures.
Molecular Profiling
Furthermore, characterization techniques include genomic, transcriptomic, and proteomic analyses. These approaches provide a detailed understanding of the molecular landscape of cultured cells. By analyzing gene expression, protein production, and other molecular features, researchers can uncover insights into cell function and regulation. This multi-dimensional analysis enhances the overall understanding of cellular processes and disease mechanisms.
Conclusion: Importance of Cell Characterization
In summary, the characterization of cultured cells is a fundamental practice in cell biology research. It ensures the accuracy, purity, and functionality of cell lines, thereby supporting the reliability of experimental results. By incorporating thorough characterization protocols, researchers can achieve more meaningful and reproducible outcomes, ultimately advancing scientific knowledge and medical innovation.