Culture Vessels:

In the tissue culture technology, the cells attach to the surface of a vessel which serves as the
substrate, and grow. Hence there is a lot of importance attached to the nature of the materials
used and the quality of the culture vessels. The term anchorage dependent cells are used when
the cells require an attachment for their growth. On the other hand, some cells undergo
transformation, and become anchorage independent.
Materials used for culture vessels: 1. Glass
Although glass was the original substrate used for culturing, its use is almost discontinued now.
This is mainly because of the availability of more suitable and alternate substrates.
2. Disposable plastics:
Synthetic plastic materials with good consistency and optical properties are now in use to
provide uniform and reproducible cultures. The most commonly used plastics are polystyrene,
polyvinyl chloride (PVC), polycarbonate, metinex and thermonex (TPX).
Types of culture vessels:
The following are the common types of culture vessels.
i. Multiwall plates ii. Petridishes iii. Flasks iv. Stirrer bottles.
The actual choice of selecting a culture vessel depends on several factors:
1. The way cells grow in culture—monolayer or suspension.
2. The quantity of the cells required.
3. The frequency of sampling for the desired work.
4. The purpose for which the cells are grown.
5. The cost factor.
In general, for monolayer cultures, the cell yield is almost proportional to the surface area of the
culture vessel. The flasks are usually employed for this purpose. Any type of culture vessel can
be used to grow suspension cultures. It is necessary to slowly and continuously agitate the
suspended cells in the vessel.
Treatment of culture vessel surfaces:
For improving the attachment of cells to the surfaces, and for efficient growth, some devices
have been developed. It is a common observation that the growth of the culture cells is better on
the surfaces for second seeding. This is attributed to matrix coating of the surfaces due to the
accumulation of certain compounds like collagen and fibronectin released by the cells of the
previous culture. There are now commercially available matrices (e.g. matrigel, pronectin, and
cell-tak).
Feeder layers:
Some of the tissue cultures require the support of metabolic products from living cells e.g. mouse
embryo fibroblasts. In this case, the growing fibroblasts release certain products which when fed
to new cells enhance their growth.
Alternate substrates as substitutes of culture vessels:
In recent years, certain alternatives for culture vessels have been developed. The important
alternative artificial substrates are micro carriers and metallic substrates.
Micro-carriers:
They are in bead form and are made up of collagen, gelatin, polyacrylamide and polystyrene.
Micro-carriers are mostly used for the propagation of anchorage-dependent cells in suspension.
Metallic substrates:
Certain types of cells could be successfully grown on some metallic surfaces or even on the
stainless steel discs. For instance, fibroblasts were grown on palledium.
Use of Non-Adhesive Substrates in Tissue Culture:
The growth of anchorage independent cells can be carried out by plating cells on non-adhesive
substrate like agar, agarose and methyl cellulose. In this situation, as the cell growth occurs, the
parent and daughter cells get immobilized and form a colony, although they are non-adhesive.

Sterilization:
The sterilization procedures are designed to kill the microorganisms, besides destroying the
spores.
There are three major devices for sterilization:
1. Dry heat 2. Moist heat (autoclave) 3. Filters.
In the Table 33.2, the sterilization of major equipment, apparatus and liquids is given.

Sterilization by dry heat:

This is carried out at a minimum temperature of 160°C for about one hour.
Sterilization by moist heat:
Certain fluids and perishable items can be sterilized in an autoclave at 121°C for 15-20 minutes.
For effective moist heat sterilization, it is necessary that the steam penetrates to all the parts of
the sterilizing materials.
Sterilization by filters:
The use of filters for sterilization of liquids often becomes necessary, since the constituents of
these liquids may get destroyed at higher temperatures (dry heat or moist heat). Sterile filtration
is a novel technique for heat- labile solutions. The size of micropores of the filters is 0.1-0.2 µm.
Filters, made from several materials are in use. These materials include nylon, cellulose acetate,
cellulose nitrate, polycarbonate, polyethersulfone (PES) and ceramics.

The filters are made in different designs-disc filters, cartridges and hollow fiber. In fact, many
commercial companies (e.g. Millipore, Durapore) supply reusable and disposable filters,
designed for different purposes of sterilization.

Types of cultures: 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
tothecellculture.
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
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, Single chain urokinase-type plasminogen activator
TCL-598 (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
1. Tissue-type plasminogen activator (t-PA)
2. B-and gamma interferons
Chinese hamster ovary (CHO) cells
3. Factor VIII

Characterstics of cell in culture:

1. Contact inhibition:
Inhibition of cell motility and plasma membrane ruffling when the cells are in complete contact
with other adjacent cells. This mostly occurs at confluence state, and results in the ceasation of
the cell proliferation.
2. Anchorage dependence:
Anchorage dependence refers to the need for cells to be adhered to or in contact with another
layer of cells. The cells can be adhered to other cells, extracellular matrix, or tissue culture
plastic (via proteins). In any manner, many cell types require some sort of anchorage in order to
survive. Therefore, if an anchorage dependent cell is not adhered and floating around, it will
die. The cell knows of its anchored state through physical cytoskeletal signalling, as well as
through juxtacrine or gap/tight junction mediated signalling. This is relevant to a certain type of
cell called epithelial cells. Epithelial cells make up the lining of hollow organs and glands, as
well as skin and other enclosed organs. These cells are packed on top of each other in various
ways (columnar, cuboidal, squamous etc.) As these cells ARE anchorage dependent, they will
die if they are no longer adhered to other cells - this is referred to as anoikis.

4. Cellular Senescence:
The growth of the cells is usually measured as population doublings (PDs). The PDs refer to the
number of times the cell population doubles in number during the period of culture and is
calculated by the following formula.
Log10 (No. of cells harvested) – log10 (No. of cells seeded)/ log102
The phenomenon of senescence has been mostly studied with human fibroblast cultures. After
30-60 populations doublings, the culture is mainly composed of senescent fibroblasts.
These senescent fibroblast are unable to divide in response to mitotic stimuli. It must be noted
that the cells do not appear suddenly, but they gradually accumulate and increase in number
during the life span of the culture.
The different parameters used for the measurement of cell growth in cultures are listed
below:
a. Direct measure of cell number. b. Determination of DNA/RNA content.
c. Estimation of protein/ATP concentration.
Measurement of Senescence: The direct measurement of senescent cells is rather difficult.
Some of the indirect measures are:
a. Loss of metabolic activity b. Lack of labeled precursor (3H-thymidine) incorporation into
DNA. c. Certain histochemical techniques.
Senescence-associated β-galactosidase activity assay
There occurs an overexpression of the lysosomal enzyme β-galactosidase at senescence. This
enzyme elevation is also associated with an increase in the cell size as the cell enters a permanent
non-dividing state. The number of senescent cells in a culture can be measured by senescence-
associated β-galactosidase (SA-β) assay.

Immortal cells:
An immortalized cell line is a population of cells from a multicellular organism which would
normally not proliferate indefinitely but, due to mutation, have evaded normal cellular
senescence and instead can keep undergoing division. The cells can therefore be grown for
prolonged periods in vitro. The mutations required for immortality can occur naturally or be
intentionally induced for experimental purposes. Immortal cell lines are a very important tool for
research into the biochemistry and cell biology of multicellular organisms. Immortalised cell
lines have also found uses in biotechnology.
An immortalized cell line should not be confused with stem cells, which can also divide
indefinitely, but form a normal part of the development of a multicellular organism.
Examples: There are several examples of immortalized cell lines, each with different properties.
Most immortalized cell lines are classified by the cell type they originated from or are most
similar to biologically.

 3T3 cells – a mouse fibroblast cell line derived from a spontaneous mutation in cultured
mouse embryo tissue.
 A549 cells – derived from a cancer patient lung tumor.
 HeLa cells – a widely used human cell line isolated from cervical cancer patient Henrietta
Lacks.
 HEK 293 cells – derived from human fetal cells.
 Jurkat cells – a human T lymphocyte cell line isolated from a case of leukemia.
 Vero cells – a monkey kidney cell line that arose by spontaneous immortalisation.