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Many cell lines, especially those derived from normal tissues, are considered to

be Anchorage-Dependent, that is, they can only grow when attached to a

suitable substrate. Some cell lines that are no longer considered normal
(frequently designated as Transformed Cells) are frequently able to grow
either attached to a substrate or floating free in suspension; they are
Anchorage-Independent. In addition, some normal cells, such as those found
in the blood, do not normally attach to substrates and always grow in

Basic characteristics of tissue culture

Tissue culture
Tissue culture is used as a generic term to include the in vitro cultivation of organs,
tissues and cells. Originally, the term is not limited to animal cells, but includes the in
vitro cultivation of plant cells. Tissue culture can be subdivided into three major
categories; organ culture, explant culture, and cell culture.
Organ culture
Organ culture refers to a three-dimensional culture of tissue retaining some or all of the
histological features of the tissue in vivo. The whole organ or part of the organ is
maintained in a way that allows differentiation and preservation of architecture, usually
by culturing the tissue at the liquid-gas interface on a grid or gel.
There are disadvantages to organ cultures. Organs cannot be propagated so each piece
of tissue can only be used once, which makes it difficult to assess the reproducibility of a
response. And, of course, the particular cells of interest may be very small in number in
a given piece of tissue so the response produced may be difficult to detect and quantify.
It may not be possible to supply adequate oxygen and nutrients throughout the tissue
because of the absence of a functioning vascular system, so necrosis of some cells occurs
fairly rapidly. This problem may be ameliorated to some extent by keeping the organ in
stirred cultures or in roller bottles which alternately provide air and soluble nutrients.
Explant (or organotypic) culture
In explant culture, small pieces of the tissue of interest are simply allowed to attach to an
appropriate substrate, usually one that has been coated with collagen, and are cultured
in a rich medium, usually one containing serum. Following attachment, cell migration is
promoted in the plane of the solid substrate. Traditionally, explants have been
maintained in Maximov chambers in which cells are grown on coverslips sealed over a
depression in a thick glass slide, and this approach is still in use. More recently, it has
become common to use regular culture dishes, which are much more convenient since
they do not need to be disassembled and reassembled at each feeding. As with
dissociated cell culture, immature tissue grows best, and explants are generally prepared
from embryonic or neonatal tissue. Typically, the tissue is cut with scalpels into slices 0.5
to 1.0 mm thick, but in some cases it is simply fragmented by passing through a nylon
mesh. The need for diffusion of nutrients and oxygen to the center of the explant limits
thickness to about a millimeter.
In experienced hands, explant cultures can be maintained for months, and cells within
the explant continue their development more or less appropriately. One of the principal
advantages of this method is that some aspects of the tissue's architecture can be
preserved within the explant.
(Dissociated) Cell culture
Cell culture refers to cultures derived from dissociated cells taken from the original
tissue ('primary cell culture'). Cells are dispersed (mechanically and/or enzymatically)
into a cell suspension which may then be cultured as a monolayer on a solid substrate, or
as a suspension in the culture medium. These cultures have lost their histotypic
architecture and often some of the biochemical properties associated with it. However,
they can be propagated and hence expanded and divided to give rise to replicate cultures.
Cell cultures can be characterized and a defined population can be preserved by freezing.
The most obvious advantage of cell culture, and of dissociated cell culture in particular,
is that it makes individual living cells accessible. All in all, primary dissociated cell
cultures are particularly amenable to study using morphological and physiological
techniques, which can be applied on a cell by cell basis. They are obviously less well
suited to traditional biochemical approaches because the quantity of material obtainable
from these cultures is usually limited and they contain a heterogeneous population of
One final drawback of working with primary cell cultures is that success is not automatic.
Finding the conditions that permit good cell growth and maturation, getting culture to
grow reproducibly, and documenting that you have accomplished all of this entails
plenty of hard work.

Selecting types of animal cell culture

Organ culture or cell culture
Early attempts at culturing tissues relied upon the explantation of whole tissue or organ
which could be maintained in vitro for only very short periods. Nowadays it is more
usual to grow specific cell types from tissues, although there are still some situations
where it is necessary to grow a whole organ (or a part of it).
In adopting a particular type of culture the following points should be taken into
account. Organ culture will preserve cell interaction, retain histological and biochemical
differentiation for longer, and, after the initial trauma of explantation and some central
necrosis will generally remain in a non-growing steady state for a period of several days
and even weeks. They cannot be propagated, generally incur greater experimental
variation between replicates, and tend to be more difficult to use for quantitative
determinations due to minor variations in geometry and constitution.
Adherent or suspension culture
Cells may grow as an adherent monolayer or in suspension. Adherent cells are said to be
anchorage-dependent and attachment to a substratum is a prerequisite for proliferation.
They are generally subjected to contact inhibition, which means they grow as an
adherent monolayer and stop dividing when they reach such a density that they touch
each other. Most cells, with the exception of mature hemopoietic cells and transformed
cells, grow in this way.
In contrast to anchorage-dependent cells, cells cultured from blood, spleen or bone
marrow adhere poorly if at all to the culture dish. In the body, these cells are held in
suspension or are only loosely adherent. It is important to realize this if you are working
with this category of cells, since the methods used to propagate these cells are very
different to those for adherent cells.
Suspension cultures are easier to propagate, since subculture only requires dilution with
medium. Cultures in which cells grow attached to each other or to a substratum have to
be treated by a proteolytic enzyme to break the bond between cells and substratum. The
most commonly used enzyme is trypsin (other enzymes, e.g., collagenase, papain,
dispase, and pronase, are also used). Clearly, freely suspended cultures do not require
trypsinization. They are, therefore, also easier to harvest.
Primary cultures or continuous cell lines
If you remove tissue from an embryo, dissociate it into a suspension of single cells, and
plate them out onto a culture dish, a series of characteristic events occurs. Firstly, cells
are in a lag phase, usually no more than 1-2 days in length, during which there is little or
no increase in cell number. During this time, cells are "conditioning" the medium,
undergoing internal cytoskeletal and enzyme changes and adjusting to the new medium.
Secondly, the cells undergo a period of rapid division, so-called log phase growth. Then,
as they approach confluency and form contacts with one another, their rate of division
slows and they begin to express a program of differentiation characteristic of their tissue
of origin. Muscle cells fuse and acquire cross-striation, epithelial cells from the kidney or
gut become linked by junctional complexes and transport ions from one surface to
another, heart cells begin to beat spontaneously.

The organotypic model consists of three approaches for the original structural
and functional interactive relationships of the organ.
The three approaches are: (1) Organ Cultures (2) Histotypic Cultures and (3)
Organotypic Cultures.

The cell cultures are widely used in the laboratories world over for various
purposes. In vitro studies with isolated cells are useful for understanding of many
cell functions such as transcription, translation, cell proliferation, respiration and
glycolysis. Thus for the study of biology and many functions, the cells grown in
conventional and monolayer cultures may be adequate.

However, for the study of integrated cellular functions or organ

functions, isolated cells will be not be of much use, as explained

Cellular Interactions in Organ Functions:

content is associated with the risk of O2induced toxicity e.g. nutrient metabolite
exchange is severely affected.
Growth and Differentiation:
In general, the organ cultures do not grow except some amount of proliferation
that may occur on the outer cell layers.

Advantages of Organ Cultures:

i. Provide a direct means of studying the behaviour of an integrated tissue in the

ii. Understanding of biochemical and molecular functions of an organ/tissue

becomes easy.

Limitations of Organ Cultures:

i. Organ cultures cannot be propagated, hence for each experiment there is a need
for a fresh organ from a donor.

ii. Variations are high and reproducibility is low.

iii. Difficult to prepare, besides being expensive.

Techniques of Organ Culture:

The most important requirement of organ or tissue culture is to place them at
such a location so that optimal nutrient and gas exchanges occur.

This is mostly achieved by keeping the tissue at gas- limited interface

of the following supports:
i. Semisolid gel of agar.
ii. Clotted plasma.
iii. Micro-porous filter.
iv. Lens paper.
v. Strip of Perspex or Plexiglas.

In recent years, filter-well inserts are in use to attain the natural geometry of
tissues more easily.

Procedure for Organ Culture:

The basic technique of organ culture consists of the following stages:
1. Dissection and collection of the organ tissue
2. Reduce the size of the tissue as desired, preferably to less than I mm in
3. Place tissue on a support at the gas medium interface.
4. Incubate in a humid CO2 incubator.
5. Change the medium (M199 or CMRL 1066) as frequently as desired.
6. The organ culture can be analysed by histology, autoradiography and

2. Histotypic Cultures:
Growth and propagation of cell lines in three- dimensional matrix to high cell
density represent histotypic cultures. The advantage with this culture system is
that dispersed monolayer cultures can be used to regenerate tissue-like
structures. The commonly used techniques in histotypic cultures use gel and
sponge hollow fibers and spheroids.

Gel and Sponge Technique:

The cells (normal or tumor) in culture can penetrate gels (collagen) or sponges
(gelatin) which provides a matrix for morphogenesis of primitive cells. This
approach has been used for the development of mammary epithelium, and some
tubular and glandular structures.

Hollow Fibers Technique:

In recent years, perfusion chambers with a bed of plastic capillary fibers have
been developed. The advantage of using hollow fibers in histotypic cultures is that
nutrient and gas exchange is more efficient. As the cells attached to capillary
fibers grow, there occurs an increase in cell density to form tissue-like structures.

Many workers claim that the behaviour of high-density cells formed on hollow
fibers is comparable to their in vivo behaviour. For instance, choriocarcinoma
cells grown in hollow fiber cultures release more chorionic gonadotrophin than in
a conventional monolayer. Hollow fiber culture techniques are regarded as ideal
systems for the industrial production of several biologically important
compounds. Work is progressing in this direction.

3. Organotypic Cultures:
Organotypic culture basically involves the combination of cells from different
lineages in a determined ratio to create a component of an organ. With the
advances in the organotypic culture techniques, it is now possible to develop
certain tissues or tissue models.

i. Skin equivalents have been created by co-culturing dermis with epidermis with
interviewing layers of collagen.

ii. Models for prostate and breast.

iii. Models for control of growth and differentiation of lung.

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.