Cell Culture Techniques - Basic Principles

Cell Culture Techniques - Basic Principles

• Cell culture is the removal of cells from an organism and their growth in a favorable artificial environment.
• Organisms used in cell culture include molluscs, fish, rabbits, humans, monkeys, insects, and plants, as well as others.
• Cell culture is used in research (basic, toxicity, drug screening, disease, genetic engineering, and gene therapy), regulatory testing purposes, and industry (protein synthesis, vaccines, tissue/organ replacement, and cellular agriculture).
• Examples of applications in toxicology include studies on heavy metals toxicity and nano-toxicity induced by TiO2 and ZnO.
• Fish cell lines are used in in vitro toxicity testing and to study toxicology pathways.
• Cell lines are used for fish health management (disease diagnosis, safety, nutrition), and research on other challenging aspects of aquaculture.
• Unique collagen fibers are used for biomedical applications. Specific compounds from marine sponge-associated fungi are researched.
• A variety of fish cell lines are available and used in research applications.

How Cells are Obtained

• Primary cultures are directly obtained from the original tissue/organ.
• Cells can be obtained from explant cultures, organ cultures, and organotypic cultures.
• Disaggregated tissue can be used in cell culture, resulting in monolayer formation.

Cell Culture Types

• Primary culture - Cells are obtained from tissue/organ; have a limited number of cell divisions.
• Cell lines - Derived from primary cultures; immortalized (unlimited divisions) or finite (limited divisions).

Primary Culture from Fish

• Fish are anesthetized to isolate cells of interest.
• Liver, gills, and kidneys are common sources for primary cells.
• Cells are isolated using enzymatic solutions, then washed.
• Cell viability is determined.

Cell Immortalization

• This allows cells to divide infinitely.
• It is done through spontaneous mutation or virus infection.
• Some cell lines express Telomerase Reverse Transcriptase (TERT) Protein.

Cell Growth

• Primary cultures have a limited number of cell divisions.
• Finite cell lines also have a limited lifespan, but will divide more times than primary cultures.
• Various cell lines are available for different applications, based on their specific properties (speed, efficiency, cost).

Primary Culture vs Cell Lines

• Primary culture - Retains many differentiated characteristics; higher degree of specificity; more labor intensive.
• Cell lines - Propagated indefinitely; more homogenous in cell type; readily available.

Type of growth

• Adherent cultures - Cells grow attached to a surface.
• Suspension cultures - Cells float freely in the culture medium.

Cell Culture Morphology

• Fibroblastic cells have elongated shapes and grow attached to surfaces.
• Epithelial-like cells are polygonal and grow attached to substrates.
• Lymphoblast-like cells are spherical and usually grow in suspension.

Cell Culture Outputs

• Molecular Biology (gene expression, protein expression)
• Flow Cytometry
• Microscopy
• Spectroscopy
• Cytogenetics

Cell Culture Contaminations

• Chemical contamination - Impurities/toxins in media, serum, and water.
• Biological contamination - Bacteria, mycoplasma, virus, fungi, yeast.
• Cross contamination - Contamination between multiple cell lines in same culture medium.

Cell Culture Authentication

• Methods to confirm identity/origin.
• Karyotyping, Isoenzyme analysis, STR and SNP analysis are used.
• Crucial when using cell cultures for research.

Aseptic Technique

• Goal: to prevent microbial contamination.
• Essential for maintaining high-quality cell culture.
• Includes sterile work areas, reagents, media; proper personal hygiene and handling.

Maintenance of Cell Cultures

• Changing media: maintaining growth conditions.
• Subcultivation: allow propagation.
• Density/Viability tests are done to maintain healthy cell growth
• Freezing/thawing allows for storage of cells.

Subculturing

• Transfer of cells from a previous culture to fresh growth medium for propagation.
• Allows the creation of new cultures from existing cell lines.

Cryopreservation

• Freezing cells for long-term storage.
• Slow/fast cooling rates to minimize risk of ice crystal damage.
• Use of freezing media (DMSO).
• Thawing process to revive.

Co-Culture Models

• Two or more different cell types grown together.
• Mimics complex cellular environments in tissues.
• Useful to study interactions & cell-to-cell communication

3D Cell Culture Technology

• Methods for culturing cells in 3D environments (scaffold-free or scaffold-based).
• This mimics in vivo conditions better than 2D models.

Tumor Microenvironment

• Components that form the environment surrounding a tumor (e.g., immune cells, extracellular matrix, blood vessels).
• 3D models are important to study effects of surrounding tissue(s) on tumor cells.

Cell Culture - 2D vs 3D

• 2D: flat surface, limited cell-cell contact, no gradients, simple setup.
• 3D: more complex, replicates in-vivo conditions, cell-cell contact, gradients.

Cell Culture - Organs-on-chip

• Miniature scale device mimicking organs' functions and microenvironment.
• Possible applications in fish physiology, toxicity testing.

Explore the essential principles and applications of cell culture techniques used in various fields such as research and industry. This quiz covers the basics of cell removal and growth, types of organisms involved, and specific applications in toxicology and aquaculture. Test your knowledge on the importance of cell culture in modern science.