Growing Cells in Culture
Part 1: Terminology
Cell culture:
The maintenance of cells outside of the living animal ( in vitro) for easier experimental
manipulation and regulation of controls .
Pros :
Use of animals reduced
Cells from one cell line are homogenous and have same growth requirements, enhancing
growing patterns.
In vitro models allow for control of the extracellular environment .
Able to monitor various elements and secretions without interference from other
biological molecules that occurs in vivo .
Cons :
Use of animals reduced
Cells from one cell line are homogenous and have same growth requirements, enhancing
growing patterns.
In vitro models allow for control of the extracellular environment .
Able to monitor various elements and secretions without
interference from other biological molecules that occurs in vivo .
Classification of Cell Cultures :
Primary Culture: Cells taken directly from a tissue to a dish .
Sub culturing: Subculture ( or ( passage) refers to the transfer of cells from one culture vessel to
another culture vessel. This is periodically required to provide fresh nutrients and growing space
for continuously growing cell lines .
The process involves removing the growth media and disassociating the adhered cells ( usually
enzymatically). Such cultures may be called secondary cultures .
Through Passaging daughter cultures are formed which represents the beginnings of a cell line .
1
,Passage number: The number of times the cells have been removed ( or" split") from the plate
and re- plated .Always write this on your plate or flask as" #P "
Cell Line: A cell line or cell strain may be finite or continuous depending upon whЕТher it has
limited culture life span or it is immortal in culture. On the basis of the life span of culture, the cell
lines are categorized into two types :
1- Finite cell lines- The cell lines which have a limited life span and go through a limited
number of cell generations ( usually 20-80 population doublings) are known as finite cell
lines, These cell lines exhibit the property of contact inhibition, density limitation and
anchorage dependence. The growth rate is slow and doubling time is around 24-96 hours .
2- Continuous cell lines- Cell lines transformed under laboratory conditions or in vitro culture
conditions give rise to continuous cell lines. These cell lines show absence of contact
inhibition and anchorage dependence. They grow either in a monolayer or in suspension (
see below). The growth rate is rapid and doubling time can be 12-24 hours.
2
, Part 2: Understanding Cell Behavior
Contact Inhibition :
When cells contact each other, they cease their growth .
Cells arrest in GO phase of the cell cycle( resting phase (
Transformed cells will continue to proliferate and pile upon each other
Anchorage Dependence
Cells that attach to surfaces in vivo require a surface to attach to in vitro .
Other cells or specially treated plastic or other biologically active coatings .
Blood cells are primary exception ( Anchorage independent).
Monolayer culture:
the cells will attach to the substrate and that normally the cells will be propagated in this
mode. These cells are Anchorage dependence means that attachment to the substrate is a
prerequisite for cell proliferation. Monolayer culture is the mode of culture common to
most normal cells, with the exception of hematopoietic cells.
Suspension cultures:
are derived from cells that can survive and proliferate without attachment These cells are
Anchorage independent this ability is restricted to hematopoietic cells, transformed cell
lines, and cells from malignant tumors .
Confluency:
how" covered" the growing surface appears
Optimal confluency:
for moving cells to a new dish is 70-80%
Too low, cells will be in lag phase and won't proliferate
Too high, cells may undergo unfavorable changes and will be difficult to remove from
plate .
3
Part 1: Terminology
Cell culture:
The maintenance of cells outside of the living animal ( in vitro) for easier experimental
manipulation and regulation of controls .
Pros :
Use of animals reduced
Cells from one cell line are homogenous and have same growth requirements, enhancing
growing patterns.
In vitro models allow for control of the extracellular environment .
Able to monitor various elements and secretions without interference from other
biological molecules that occurs in vivo .
Cons :
Use of animals reduced
Cells from one cell line are homogenous and have same growth requirements, enhancing
growing patterns.
In vitro models allow for control of the extracellular environment .
Able to monitor various elements and secretions without
interference from other biological molecules that occurs in vivo .
Classification of Cell Cultures :
Primary Culture: Cells taken directly from a tissue to a dish .
Sub culturing: Subculture ( or ( passage) refers to the transfer of cells from one culture vessel to
another culture vessel. This is periodically required to provide fresh nutrients and growing space
for continuously growing cell lines .
The process involves removing the growth media and disassociating the adhered cells ( usually
enzymatically). Such cultures may be called secondary cultures .
Through Passaging daughter cultures are formed which represents the beginnings of a cell line .
1
,Passage number: The number of times the cells have been removed ( or" split") from the plate
and re- plated .Always write this on your plate or flask as" #P "
Cell Line: A cell line or cell strain may be finite or continuous depending upon whЕТher it has
limited culture life span or it is immortal in culture. On the basis of the life span of culture, the cell
lines are categorized into two types :
1- Finite cell lines- The cell lines which have a limited life span and go through a limited
number of cell generations ( usually 20-80 population doublings) are known as finite cell
lines, These cell lines exhibit the property of contact inhibition, density limitation and
anchorage dependence. The growth rate is slow and doubling time is around 24-96 hours .
2- Continuous cell lines- Cell lines transformed under laboratory conditions or in vitro culture
conditions give rise to continuous cell lines. These cell lines show absence of contact
inhibition and anchorage dependence. They grow either in a monolayer or in suspension (
see below). The growth rate is rapid and doubling time can be 12-24 hours.
2
, Part 2: Understanding Cell Behavior
Contact Inhibition :
When cells contact each other, they cease their growth .
Cells arrest in GO phase of the cell cycle( resting phase (
Transformed cells will continue to proliferate and pile upon each other
Anchorage Dependence
Cells that attach to surfaces in vivo require a surface to attach to in vitro .
Other cells or specially treated plastic or other biologically active coatings .
Blood cells are primary exception ( Anchorage independent).
Monolayer culture:
the cells will attach to the substrate and that normally the cells will be propagated in this
mode. These cells are Anchorage dependence means that attachment to the substrate is a
prerequisite for cell proliferation. Monolayer culture is the mode of culture common to
most normal cells, with the exception of hematopoietic cells.
Suspension cultures:
are derived from cells that can survive and proliferate without attachment These cells are
Anchorage independent this ability is restricted to hematopoietic cells, transformed cell
lines, and cells from malignant tumors .
Confluency:
how" covered" the growing surface appears
Optimal confluency:
for moving cells to a new dish is 70-80%
Too low, cells will be in lag phase and won't proliferate
Too high, cells may undergo unfavorable changes and will be difficult to remove from
plate .
3
