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Characteristics of Each Culture Type

The majority of the cells derived from vertebrates, with the exception of hematopoietic cell lines and a
few others, are anchorage-dependent and have to be cultured on a suitable substrate that is specifically
treated to allow cell adhesion and spreading (i.e., tissue-culture treated). However, many cell lines can
also be adapted for suspension culture. Similarly, most of the commercially available insect cell lines
grow well in monolayer or suspension culture.

Cells that are cultured in suspension can be maintained in culture flasks that are not tissue-culture treated,
but as the culture volume to surface area is increased beyond which adequate gas exchange is hindered
(usually 0.2 0.5 mL/cm2), the medium requires agitation. This agitation is usually achieved with a
magnetic stirrer or rotating spinner flasks.

Adherent Cell Culture Suspension Cell Culture

Appropriate for most cell types, Appropriate for cells adapted to

including primary cultures suspension culture and a few other cell
lines that are nonadhesive (e.g.,

Requires periodic passaging, but allows Easier to passage, but requires daily cell
easy visual inspection under inverted counts and viability determination to
microscope follow growth patterns; culture can be
diluted to stimulate growth

Cells are dissociated enzymatically (e.g., Does not require enzymatic or

TrypLE Express, trypsin) or mechanical dissociation

Growth is limited by surface area, which Growth is limited by concentration of

may limit product yields cells in the medium, which allows easy

Requires tissue-culture treated vessel Can be maintained in culture vessels that

are not tissue-culture treated, but
requires agitation (i.e., shaking or
stirring) for adequate gas exchange

Used for cytology, harvesting products Used for bulk protein production, batch
continuously, and many research harvesting, and many research
applications applications

Primary cell culture

The maintenance of growth of cells dissociated from the parental tissue (such as kidney, liver) using the
mechanical or enzymatic methods, in culture medium using suitable glass or plastic containers is called Primary
Cell Culture.

The primary cell culture could be of two types depending upon the kind of cells in culture.

a) Anchorage Dependent /Adherent cells- Cells shown to require attachment for growth are set to be
Anchorage Dependent cells. The Adherent cells are usually derived from tissues of organs such as kidney where
they are immobile and embedded in connective tissue. They grow adhering to the cell culture.

b) Suspension Culture/Anchorage Independent cells - Cells which do not require attachment for growth or do
not attach to the surface of the culture vessels are anchorage independent cells/suspension cells. All suspension
cultures are derived from cells of the blood system because these cells are also suspended in plasma in vitro e.g.

Secondary cell cultures

When a primary culture is sub-cultured, it becomes known as secondary culture or cell line. Subculture (or
passage) refers to the transfer of cells from one culture vessel to another culture vessel.

Subculturing- Subculturing or splitting cells is required to periodically provide fresh nutrients and growing space for
continuously growing cell lines. The process involves removing the growth media, washing the plate, disassociating
the adhered cells, usually enzymatically. Such cultures may be called secondary cultures.

Cell Line

A Cell Line or Cell Strain may be finite or continuous depending upon whether 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:

a) 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.
b) Continuous Cell Lines - Cell lines transformed under laboratory conditions or in vitro culture conditions give
rise to continuous cell lines. The cell lines show the property of ploidy (aneupliody or heteroploidy), absence of
contact inhibition and anchorage dependence. They grow in monolayer or suspension form. The growth rate is
rapid and doubling time is 12-24 hours.

c) Monolayer cultures - When the bottom of the culture vessel is covered with a continuous layer of cells, usually
one cell in thickness, they are referred to as monolayer cultures.

d) Suspension cultures - Majority of continuous cell lines grow as monolayers. Some of the cells which are non-
adhesive e.g. cells of leukemia or certain cells which can be mechanically kept in suspension, can be propagated in
suspension. There are certain advantages in propagation of cells by suspension culture method.

These advantages are:

(a) The process of propagation is much faster.,
(b) The frequent replacement of the medium is not required.,
(c) Suspension cultures have a short lag period,
(d) treatment with trypsin is not required,
(e) a homogenous suspension of cells is obtained,
(f) the maintenance of suspension cultures is easy and bulk production of the cells is easily achieved.,
(g) scale-up is also very convenient.

The cell lines are known by:

a) A code e.g. NHB for Normal Human Brain.

b) A cell line number- This is applicable when several cell lines are derived from the same cell culture source e.g.
c) Number of population doublings, the cell line has already undergone e.g. NHB2/2 means two doublings.



The cell lines are characterized by their a) growth rate and b) karyotyping.

a) Growth Rate - A growth curve of a particular cell line is established taking into consideration the population
doubling time, a lag time, and a saturation density of a particular cell line. A growth curve consist of:

1) Lag Phase: The time the cell population takes to recover from such sub culture, attach to the culture vessel and

2) Log Phase: In this phase the cell number begins to increase exponentially.
3) Plateau Phase: During this phase, the growth rate slows or stops due to exhaustion of growth medium or

b) Karyotyping - Karyotyping is important as it determines the species of origin and determine the extent of gross
chromosomal changes in the line. The cell lines with abnormal karyotype are also used if they continue to perform
normal function. Karyotype is affected by the growth conditions used, the way in which the cells are subcultured
and whether or not the cells are frozen.

c) There are certain terms that are associated with the cell lines.

These are as follows:

(i) Split ratio- The divisor of the dilution ratio of a cell culture at subculture.
(ii) Passage number- It is the number of times that the culture has been cultured.,
(iii) Generation number- It refers to the number of doublings that a cell population has undergone.