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Saturday, July 19, 2008

Animal Cell Culture

Animal cell culture (ACC) is the process of culture of animal cells outside the tissue (ex vivo) from which they were obtained. The process of ACC is carried out under strict laboratory conditions of asepsis, sterility and controlled environment involving temperature, gases and pressure. It should mimic the in vivo environment successfully such that the cells are capable of survival and proliferation in a controlled manner.

Culturable Cells:
Theoretically, cells of any type can be cultured upon procurement in a viable state from any organ or tissue. However, not all types of cells are capable of survival in such an artificial environment because of many reasons on which the artificial environment may fail to mimic the biochemical parameters of the source environment. Some good examples include the absence of Growth Regulators, cell-to-cell signal molecules, etc. Under optimal conditions of maintenance, the cell culture established can be sub-until a of specific cell type is obtained. This can repeatedly sub-cultured to maintain as a cell-line. As a matter of fact, cell lines from cancerous tissues have also been established. The presence of excess growth regulators or other factors may often render the cells to undergo rapid uncontrolled proliferation resulting in a cancerous state. Good examples of established cell lines are Hela, BHK,Vero and CHO etc.

Important Terms:
Confluent – crowding of cell substrate such that there is no space for cells to grow.

Passage/subculture – the transfer of cell with or without dilution, from one culture vessel to another.

Passage Number – the number of times the cells in the culture have been subcultured or passaged.


Anchorage dependent: cells or cultures derived from them, which will grow, survive, or maintain  function only when attached to a surface such as glass or plastic.

Suspension culture: a type of culture in which cells, or aggregates of cells, multiply while suspended in a liquid medium.


Cell Growth & Propagation :
·
Monolayer Cultures:


Cell Culture in T-Flask - Image Source


1. Anchorage dependent cultures:

Advantages: -Easy to change medium and wash cells before adding fresh medium.
- Microscopic evaluations
- Homogenous distribution

Disadvantages: – Scale up is difficult and expensive
- Require more space than suspension cultures
Properties: – Confluency
- Pretreated surfaces for growth

2. Suspension cultures:


Advantages: Trypsin treatment not required, so subculture is quicker and less traumatic for the cells.
- Scale up is easier

Disadvantages: Prolonged use of same vessel can lead to contamination
Properties: Untreated vessels used, minimise cost
- kept in suspension mechanically

Culture on Microcarriers :
· bead like structures used (90-250µm dia) which provide a large surface area to cells made from cellulose,sephadex.
· cells attach and spread on beads, grow to a confluent monolayer.
· held in medium and stirred constantly
· combination of suspension and adherent technique.


Suspension Culture- Image Source

Cell Growth in Media

The proliferation of anchorage dependent cells can only occur after adhesion to a suitable culture surface. Extracellular surface molecules are found naturally in some media supplements and are also secreted by cells themselves. ECM (extracellular matrix) molecules are adsorbed onto the substrate surface and then cells bind to the matrix through cell surface receptors.

Components of cell culture media :
· water
· inorganic salts
· trace elements
· sugars
· amino acids
· vitamins
· fatty acids & lipids
· hormones & growth factors
· carriers  ECM proteins
· antibiotics

Serum is an important component of most media formulations because it:
Ø is a universal growth supplement; complex mixture of components and absolutely essential for cell growth & proliferation.
Ø increases viscosity of medium thereby protecting cells from shear.
Ø buffers the cell culture environment
Ø binds and neutralises toxins( heavy metal ions and endotoxins)