Cell Culture Flashcards

Cell Culture Definition

• Cell culture is the removal of cells (animal or plant) and growth in a favorable artificial environment (in vitro).

• Cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation.

• Can be derived from a cell line that has already been already established.

• Primary Culture: Refers to the stage of the culture after the cells are isolated from the tissue and proliferated under the appropriate conditions until they occupy all of the available substrate.

• Cells have to be subcultured (i.e., passaged) transferring them to a new growth medium to provide more room for continued growth.

Cell Lines

• Passaging or subcultivation of a primary culture begins a cell line.

• Normally have a limited life span and as they are passaged, cells with the highest growth capacity predominate, resulting in a degree of genotypic and phenotypic uniformity in the population.

• An established cell culture can be made to proliferate indefinitely given the right treatment, conditions, and space (Immortalisation).

• Cell strain: Subpopulation of a cell line that has been positively selected from the culture.

Survival needs

• Survival needs according to Maslow

Culture Media

• Creates the medium (gel or liquid) to support cellular growth in an artificial environment so cells cultures can survive and proliferate.

• Cell culture media are comprised of a combination of:

  • Amino acids: a mixture of amino acids, the building blocks of protein.

  • Vitamins: facilitate cellular growth and proliferation.

  • Carbohydrates: energy source for living cells (Glucose).

  • Inorganic salts: regulate membrane potential and osmolality.

  • Basic and trace elements: iron, potassium, magnesium, and zinc needed to grow.

  • Serum: growth factors, hormones needed to influence cell function.

  • Buffering systems: regulate pH.

  • Antibiotics: inhibit fungal and bacterial growth.

Use of Cell Cultures

• Provides excellent model systems for studying the normal physiology and biochemistry of cells (e.g., metabolic studies, aging).

• Studying the effects of drugs and toxic compounds on the cells, and mutagenesis and carcinogenesis.

• Drug screening and development

• Large scale manufacturing of biological compounds (e.g., vaccines, therapeutics).

• The major advantage:

  • Consistency and reproducibility of results.

  • Study of cell behavior without the variations.

  • Control of the growth environment leads to uniformity of sample.

  • Avoids the legal, moral, and ethical problems

Cell Culture Hood Layout

• Large enough to be used by one person at a time, be easily cleanable inside and outside, have adequate lighting, and be comfortable to use without requiring awkward positions.

• Keep the work space in the cell culture hood clean and uncluttered, and keep everything in direct line of sight.

  • Disinfect each item placed in the cell culture hood by spraying them with 70% ethanol and wiping clean.

  • A wide, clear work space in the center with your cell culture flasks.

  • Pipettor where it can be reached easily.

  • Reagents and media in the rear right to allow easy pipetting.

  • Tube rack in the rear middle holding additional reagents.

  • Small container in the rear left to hold liquid waste/ outside hood for larger waste

Cell Culture Equipment

• Incubator (humid CO2 incubator recommended)

• Water bath

• Centrifuge

• Refrigerator and freezer (–20°C)

• Cell counter (e.g., Countess® Automated Cell Counter or hemacytometer)

• Inverted microscope

• Liquid nitrogen (N2) freezer or cryostorage container

• Sterilizer (i.e., autoclave)