Cell Biology & Tissue Culture: Culture Environment and Ethics

Flashcards covering the culture environment, media components, critical requirements for mammalian cell growth, and relevant ethical case studies.

Aim of Mammalian Cell Culture

To provide an environment that mimics, to the greatest extent possible, the in vivo environment of a specific cell type.

Essential Culture Requirements

Appropriate temperature, pH, oxygen, $CO_2$ supply, surface for attachment, nutrient and vitamin supply, protection from toxic agents, and specific hormones and growth factors.

HeLa

A human cell line derived from ovarian cancer.

MCF-7

A human cell line derived from breast cancer.

Vero

An African green monkey Chlorocebus kidney epithelial cell line initiated in 1962.

CHO

Chinese hamster ovary cell line.

ZF4 and AB9

Zebrafish cell lines.

Culture pH

Usually neutral, visually assessed using phenol red.

Culture Temperature

$37^{\circ}C$, maintained by an incubator.

Culture Atmosphere

$5\%$ $CO_2$, maintained by an incubator.

HEPES

A chemical buffering agent that maintains a physiological pH despite changes in carbon dioxide concentration and protects enzyme structure and function.

Osmotic Pressure & Osmolarity

Maintained as isotonic by media and PBS.

DMEM

Dulbecco's Modified Eagle's Medium, a basal and full defined media.

Role of Vitamins in Media

Most contribute to the functioning of enzymes and act as a co-factor or facilitate the production of co-factors.

Riboflavin

Essential to the formation of coenzymes FMN and FAD, involved in energy metabolism, cell respiration, antibody production, and growth.

FBS (Fetal Bovine Serum)

A media supplement and meat industry by-product containing essential growth factors; it is batch dependent and expensive.

FBS Collection Process

A needle is inserted between the ribs of a cow fetus (at least three months old) directly into the heart to vacuum blood into a sterile bag at an abattoir.

Growth Factors

Unique cell signaling molecules playing important roles in cell proliferation and development, such as FGF (Fibroblast Growth Factor) and EGF (Epidermal Growth Factor).

Serum Free Media (SFM) Advantages

Easier purification, precise evaluation of cellular function, increased definition, and more consistent performance.

Glutamine

An unstable amino acid supplement needed in cell culture to support growth and function.

Osmotic Shock

The rupture of cells in a solution following a sudden reduction in osmotic pressure.

Phenol Red: Yellow/Orange

Indicates acidic pH, meaning media is exhausted of nutrients, bacterial contamination is present, or cell overgrowth has occurred.

Phenol Red: Dark Pink/Red

Indicates basic pH, suggesting dissolved gases (CO2) are not at the correct balance ($5-10\%$).

Certificate of Analysis

A document that reports and certifies the test results of a product.

Substrate

A material providing a surface for adherent cell cultures to attach and grow on, mimicking their natural environment.

Adherent vs. Suspension Cultures

Adherent cells require a surface to form a monolayer, while suspension cells grow freely floating.