SUBCULTURE

AND
CELL LINES


SUBCULTURE AND
PRPOGATION
Once a primary culture is subcultured or passaged or transformed , it becomes known as a cell line .

This term implies the presence of several cell lineage of either similar or distinct phenotypes.

CELL STRAIN: If one cell lineage is selected by cloning or by physical cell seperation or by other techniques to have
certain specific properties that have been identified in the bulk of the cells in the culture this cell lines are called cel l
strain
Cell lines may be

FINITE CELL LINES: 20 to 80 population doubling

CONTINUOUS CELL LINES:100 to 200 population doubling


PASSAGE NUMBER: The number of times that the culture has been subcultured

GENERATION NUMBER: The number of doubling the cell population has undergone.

When the split ratio is 1:2 the passage number is approximately equal to the generation number .
For split ratio greater then 1:2 it is useful to use a power of 2 , e.g. 1:2, 1:4, 1:8 to give the equivalence of one ,two ,
three population doubling respectively neglecting the losses due to
Necrosis
Apoptosis
Differentiation
Premature ageing



CELL LINE DESIGNATION

CELL LINE CODE: e.g.
NHB : NORMAL HUMAN BRAIN

A CELL LINE NUMBER : IN CASE SEVERAL LINE ARE DERIVED FROM THE SAME SOURCE.
E.G: NHB1, NHB2 etc


The number may represent the accession number
e.g. LT156

For finite cell lines the no of population doubling is indicated after a slash

e.g. NHB2/2




For continuous cell lines the number after a slash may be used to represent the number of passage

With reference to the publication or reports each cell lines should be prefixed with a code for the laboratory in which it
was derived .

e.g. NCI for national cancer institute

ATTC for American type cell culture collection

SELECTION OF CELL LINE

FINITE VS CONTINUOS :

NORMAL OR TRANSFORMED

SPECIES

GROWTH CHARACTERISTICS

Population doubling time
Saturation density
Cloning efficiency
Ability to grow in suspension
Growth fraction

AVAILABILIBITY

VALIDATION

PHENOTYPIC EXPRESSION

STABILITY

Routine maintenance
Cell morphology: examine regularly
To conform the absence of contamination
The healthy status of the cells
The lack of signs of detoriation (granulation around the nucleus,vaculation,and
rounding of the cells with detachment from the substrate.).
Such signs may indicate : inadequate or toxic medium or serum , microbial
contamination, or senescence of the cell .

REPLACEMENT OF THE MEDIUM
1. A drop in pH:
Stops growing between pH range: 7.0 to 6.5.
2. Cell concentration:
High concentration exhaust the medium faster.
3. Cell type:
Normal cell at high cell density usually stop growing due to G1 arrest but
deteriorate little
Transformed , continuous cell lines and some embryonic cells deteriorate rapidlly at
high cell density.

4. Morphological detoriation






VOLUME ,DEPTH AND SURFACE AREA:
Usual ratio of volume :surface area is 0.2-0.5 ml/cm
2
Optimum ratio depends on the rate of gaseous diffusion
and the metabolic rate of the cell.
HOLDING MEDIUM:

Are regular media with the serum concentration reduced to
0.5%or 2% or completely eliminated.
For serum free media the growth factor and mitogens are
eliminated
This inhibits mitosis in most untransformed cells
It is used to maintain cell lines with a finite lifespan without
using up the limited number of cell generation available to
them.






SUBCULTURE:

The growth of cells
follow a standard
pattern:
Early lag phase
Middle log phase
Late log phase
Plateau phase
Death or apoptosis

Subculture involves removal of
medium and dissociation of cell with
Trypsin,alone or with other proteases
(pronase,dispase, collagenase)
Shaking the bottle
EDTA chelates ca ions thus helps dissociation
of ca dependent cadherins.











CRITERIA FOR SUBCULTURE


1. Density of culture
2. Exhaustion of medium
3. Time since last culture:
Routine subculture is best performed
according to strict schedule so that
reproducible behavior is achieved and
monitored.

4. Requirement for other procedures


Split ratios and growth cycle
When handling the cell line for the first time it is
good to practice subculture to a split ratio of 2 or 4
After gaining experiences it may be increased
accordingly.

Cell concentration at subculture:
The ideal method of determining the correct seeding
density is to perform a growth curve at different seeding
density and determine the correct minimum concentration
that gives a short lag period and early entry in rapid
logarithmic growth but reaches the plateau phase at a time
convenient for next subculture.


Propagation in suspension:



Subculture in suspension culture can be done as with bacteria and yeast.
Trypsin treatment not required
Quicker
Less traumatic
Scale up easier

Replacement of medium is not usually carried out instead the culture is
either diluted and expanded or
Some cell suspension is withdrawn and the residue is diluted back to an
appropriate seeding concentration.




criteria
Cell concentration:110
6
/ml
pH
Time since last subculture
Cell production requirement