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. lymphocytes.
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. NHB1, NHB2.
c) Number of population doublings, the cell line has already undergone e.g. NHB2/2 means two
doublings.

FIG SHOWING THE SALIENT FEATURES OF CELL CULTURE WITH EVOLUTION OF

A CELL LINE

CHARACHTERIZATION OF CELL LINES

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 spread.

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 confluency.
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.
TABLE-SOME ANIMAL CELL LINES AND THE PRODUCTS OBTAINED FROM THEM

Cell line

Product

Human tumour

Angiogenic factor

Human leucocytes

Interferon

Mouse fibroblasts

Interferon

Human Kidney

Urokinase

Transformed human kidney

cell line, TCL-598

Single chain urokinase-type

plasminogen activator (scu-PA)

Human kidney cell (293)

Human protein (HPC)

Dog kidney

Canine distemper vaccine

Cow kidney

Foot and Mouth disease (FMD) vaccine

Chick embryo fluid

Vaccines for influenza, measles and

mumps

Duck embryo fluid

Vaccines for rabies and rubella

Chinese hamster ovary (CHO)

cells

1. Tissue-type plasminogen
activator (t-PA)
2. B-and gamma interferons

3. Factor VIII