Al-Farabi University College Biotechnology

Department of Biology 2020/2019

fourth stage

Plant and Animal tissue culture

‫اعداد الطالبة‬
‫نريمان حسن شعبان علي‬
C_‫شعبة‬

‫اشراف‬

‫م نورهان صبيح‬.‫م‬

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 Plant Tissue Culture

Introduction
Tissue culture is the art of growing cells outside a living body. As we have
already discussed the historical background and current innovations of the
field of biotechnology in the first two volumes, it is well understood that there
are direct and indirect relationships between the developmental biology of
plant and animal cells [1]. There are certain challenges involved in culturing
both plant and animal cells, such as exhaustion of nutrients in the growth
medium, apoptotic/necrotic cell accumulation, cell cycle arrest (or senescence)
due to intercellular communication or contact inhibition, etc, which should be
investigated further [1]. Various approaches can be utilized to manipulate cell
cultures of both plant and animal cells. Subculturing is a common practice
which is adopted to replace old medium with new, nutrient enriched, medium.
Subculture can also be used to prevent the major problem of senescence. This
involves transferring a small number of cells into a new culture dish. The
animal–plant co-culture system has not been explored due to its greater
vulnerability to contamination of the culture. There is, however, scope to
maintain suitable aseptic conditions and encourage a co-culture system to
further study the impacts of their growth on each other [1].
The success of animal tissue culture products depends on their efficacy, cost
effectiveness and the potential for scale-up. Recent and current advances in
tissue culture science have enhanced the complexity in the design of
biomaterials which have either been proposed or utilized to grow animal cells
[2]. This complexity generally increases the difficulty for manufacturing
industries in designing suitable fabrication techniques. It is worth noting that
most of the features that are suitable for designing biomaterials originate in
the structure and function of plants [2]. Several investigations have shown that
decellularized plant tissues can be utilized as a suitable scaffold for the culture
of human cells. It has been observed that through an approach of simple
biofunctionalization it is possible to achieve the adhesion of human cells on
various sets of plant tissues. The increased water transport efficiency
and hydrophilicity of plant tissues facilitate increases in cell number over
prolonged periods of culture [2]. In addition, animal cells are able to adapt well
to the microstructure of plant frameworks without breaking any physiological
conditions.
This results in perfect cell positioning and formation of a perfect pattern over
the feeding layer of plant cells. This supportive plant tissue based micro-
framework can be utilized as an alternative potential scaffold for mammalian
cells [2].

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 Plant tissue culture
1.Definition
Plant tissue culture (PTC) is a set of techniques for the aseptic culture of
cells tissues, organs and their components under defined physical and
chemical conditions in vitro and controlled environment (Fig 1). PTC
technology also explores conditions that promote cell division and genetic
re-programming in in vitro conditions and it is considered an important tool
in both basic and applied studies, as well as in commercial application (1 )
Today, facilities for in vitro cell cultures are found in practically each plant
biology laboratory, serving different purposes because tissue culture has
turned into a basic asset for modern biotechnology, from the fundamental
biochemical aspects to the massive propagation of selected individuals.
Today five major areas, where in vitro cell cultures are being currently
applied, can be recognized: as a model system for fundamental plant cell
physiology aspects generation of genetic modified fertile individuals, large-
scale propagation of
elite materials, preservation of endangered species, and metabolic engineering
of fine chemicals.

Fig 1. A. Callus from Catharanthus roseus. B. Suspension culture from C. roseus.

C. Regeneration of plantlets from C. roseus callus. D. Tumors from C. roseus.
E. Protoplasts from C. roseus. F. Micropropagation of Agave tequilana. G. Hairy roots
from C. roseus. H. Somatic embryogenesis of Coffea canephora. I. Root culture from
C. roseus. Pictures A–E, G–I from the authors’ laboratories. Picture F from the laboratory of Dr.
Manuel Robert all of them at Centro de Investigación Cientifica de Yucatán

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2. Stages

STAGE 1: Initiation phase

The initiation phase is the first phase of tissue culture.
Here, the tissue of interest is obtained and
introduced and sterilized in order to prevent any
microorganism from negatively affecting the process.
It is during this stage that the tissue is initiated in to
culture.

STAGE 2: Multiplication stage

The multiplication phase is the second step of tissue
culture where the in vitro plant material is redivided
and then introduced in to the medium. Here, the
medium is composed of appropriate components
for growth including regulators and nutrients. These
are responsible for the proliferation of the tissue
and the production of multiple shoots.

STAGE 3: Root formation

It is at this phase that roots are formed. Here,
hormones are required in order to induce rooting,
and consequently complete plantlets.

General procedure for plant tissue culture:

Medium preparation:

1. The appropriate mixture (such as the MS mixture) is mixed with distilled

water and stirred while adding the appropriate amount of sugar and
sugar mixture. Here, sodium hydroxide or hydrochloric acid is used to
adjust the pH – Contents used here will depend on the plant to be
cultured and the number of tissues to be cultured.
2. Agar is added to the mixture, heat and stirred to dissolve.

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 3. After cooling, the warm medium is poured into polycarbonate tubes (to
a depth of about 4 cm).
4. With lids sitting on the tubes, the tubes are placed in a pressure cooker
and sterilized for 20 minutes.

Plant preparation:
1. Cut the plant part in to small pieces (about 1cm across). On the other
hand, such parts as the African violet leaves can be used as a whole.
2. Using detergent and water, wash the plant part for about 20 minutes.

3. Transfer the plant part in to sterilizing Clorox solution, shake for a

minute and leave to sock for 20 minutes.
4. Using a lid, gently discard the Clorox and retain the plant part in the
container and then cap the container.

Transferring the plant material to a tissue culture medium:

1. 70percent alcohol should be used for the sterilization of the equipment

used and containers.
2. Open the container and pour sterile water to cover half the container.
3. Cover with a sterile lid again and shake the container for 2 to 3 minutes
in order to wash the tissue and remove the bleach.
4. Pour the water and repeat this three times.
5. Using sterilized gloves, remove the plant part from the container and on
to a sterile Petri dish.
6. Using a sterile blade cut the plant material to smaller pieces of about 2
to 3 mm across avoiding the parts that have been damaged by bleach.
7. Using sterile forceps, place a section of the plant in to the medium.
8. Replace the lid/cap and close tightly.

Technique for Plant in Vitro Culture:

1. Micropropagation – This technique is used for the purposes of
developing high- quality clonal plants (a clone is a group of identical
cells). This has the potential to provide rapid and large scale propagation
of new genotypes.
2. Somatic cell genetics – Used for haploid production and somatic
hybridization
3. Transgenic plants – Used for expression of mammalian genes or plant
genes for various species it has proved beneficial for the engineering of
species that are resistant against viruses and insects.

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3.Types (especially cell line)
1. Callus
2. Suspension Cultures
3. Organ Culture
4. Protoplasts
especially cell line:-
Plants have been increasingly researched as an alternative recombinant
protein expression system. Creative Biolabs is capable of offering different
plant production systems to obtain high-quality recombinant proteins of
interest. There are three broad plant production systems: whole plant, culture
of organized plant tissues and plant cell culture. All these three systems are
able to produce recombinant proteins with complex glycosylation patterns and
post-translational modification (PTM).

Using whole plants to produce proteins has advantages that it is easy to reach
agricultural-scale production with low capital equipment costs and scalability.
But its development time is long and has variations in product yield and
quality, moreover, it is difficult to apply good manufacturing practice (GMP) to
the early stages of production. Now there are more than 100 plants that can
be induced to produce hairy roots in culture. Production of pharmaceuticals by
this system is over-production of a pharmaceutical that are most naturally
produced by the plant. The expression system of plant cell cultures avoids
these problems but retain the advantages. Firstly, plant cells can be cultured in
simple, synthetic media with inexpensive cost. Secondly, as higher eukaryotes,
they can perform PTMs that occur in human cells and synthesize complex
multimeric proteins and glycoproteins such as interleukins and
immunoglobulins. Thirdly, plant cells neither harbor human pathogens nor
produce endotoxins, so they are intrinsically safe. Finally, cGMP can be applied

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throughout the production pipeline of plant cells. Taliglucerase alfa is an
enzyme with a human compatible glycan profile which is produced by
genetically modified carrot plant root cells and has been approved by FDA.
There are various approaches that can be used for cultivation of plant cells,
such as derivation of shooty teratomas, hairy roots, suspension cells and
immobilized cells. Among which suspension cells are preferred because they
are the most amenable to GMP procedures. Up to now, suspension cells have
been identified from various plant species including Catharanthus roseus,
Taxus cuspidata, Arabidopsis thaliana and crops such as soybean, alfalfa,
tobacco, tomato, rice, etc. BY-2, NT-1 and the closely related cultivars are
tobacco suspension cells and are frequently chosen as host cell lines. These
cells have favorable growth characteristics and their transformation and
propagation are simple and well-established. During the design of constructs
used to express the recombinant proteins, promoter and lead peptide choice
affects the yield. The most widely used promoters include constitutive
promoters and inductive promoters. Cauliflower mosaic virus (CaMV) 35S,
hybrid (ocs)3mas promoter, hybrid (ocs)3mas promoter, ubiquitin promoters
from maizeand A. thaliana3, rice α-amylase RAmy3D promoter induced by
sugar deprivation are commonly used. The lead peptides are from plant or
non-plant proteins, which have equivalent function. And several human
recombinant proteins have been expressed in plant cells using their own
endogenous leaders.
Integrated by advanced platforms, Creative Biolabs performs GMP or non-GMP
process development to obtain plant cells with improving productivity.

4-Application

PTC represents a useful system for the study of the physiological, biochemical,
and molecular biology processes in plant cells. The effects of a single factor, on
a given process, can be monitoring since the culture conditions can be strictly
controlled. One of the best examples of the cell cultures’ used for such
purposes may be the study of the morphogenetic process. The conditions
provided by PTC give us an optimum system for the study of the biochemical
and molecular aspects associated with plant differentiation. Also, the response
of PTC in response to elicitation is an excellent system to study the plant cells’
response to the pathogens attack. A number of genes involved in different
aspects of such response, including those in perception of the stimulus as well
as in the signalling pathway, have been isolated and characterized in cell
cultures from different species [3].

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The changes in the membrane’s fluidity and the cellular mechanisms for
resistance to metals, salinity, or drought among others, can be analyzed
without having the interference of tissue organization [4,5]. The mechanism of
the plant cell wall biosynthesis has been widely studied using protoplast as the
main tool [6,7].
One of the fields where PTC has been most useful is the study of secondary
metabolism; the use of elicitors in cell cultures has led to the identification of
enzymes involved in the biosynthesis of different compounds [9,10]. PTChas
been the model for the study and elucidation of the purine salvage pathwayin
higher plants [11,12] as well as for the study of different aspects of nutrition of
plant cells [13].

The applications are:

1. Clonal Propagation and Micro-Propagation:
2. Biomass Energy:
3. Secondary Metabolites:
4. Genetic Variability:
5. Somatic Embryogenesis and Synthetic Seed:
6. Breaking Dormancy
7. Haploid Plants
8. Somatic Hybrids
9. Transgenic Plants
10.Germplasm Conservation

Animal cell culture

1.Definition
Animal cell culture is a significant tool for
biological research. The importance of cell
culture technology in biological science was
realized a long time ago. Earlier
dedifferentiation based experiments of cells
due to selective overgrowth of fibroblasts
resulted in the enhancement of culture
techniques. Animal cell culture involves
isolation of cells from a tissue before
establishing a culture in a suitable artificial
environment. Initial isolation of the cells
from the tissues can be achieved by disaggregation using enzymatic or
mechanical methods. The source of the isolated cells is usually an in vivo
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environment, but sometimes cells are also derived from an existing cell line or
cell strain. Animal cell culture offers suitable model systems for
investigating the following factors:
•Drug screening and development.
•Mutagenesis and carcinogenesis.
•Normal physiology and biochemistry of cells.
• Potential effects of drugs and toxic compounds on the cells.
In addition, it also permits reliable and reproducible results, and is thus
considered as a significant model system in cellular and molecular biology.

2. Stages

Growth Requirements
The culture media used for cell cultures are generally quite complex, and
culture condition widely varies for each cell type. However, media generally
include amino acids, vitamins, salts (maintain osmotic pressure), glucose, a
bicarbonate buffer system (maintains a pH between 7.2 and 7.4), growth
factors, hormones, O2 and CO2. To obtain best growth, addition of a small
amount of blood serum is usually necessary, and several antibiotics, like
penicillin and streptomycin are added to prevent bacterial contamination.
Temperature varies on the type of host cell. Most mammalian cells are
maintained at 37oC for optimal growth, while cells derived from cold-blooded
animals tolerate a wider temperature range (i.e. 15oC to 26oC). Actively
growing cells of log phage should be used which divide rapidly during culture.

Process to obtain primary cell culture

Primary cell cultures are prepared from fresh tissues. Pieces of tissues from the
organ are removed aseptically; which are usually minced with a sharp sterile
razor and dissociated by proteolytic enzymes (such as trypsin) that break apart
the intercellular cement. The obtained cell suspension is then washed with a
physiological buffer (to remove the proteolytic enzymes used). The cell
suspension is spread out on the bottom of a flat surface, such as a bottle or a
Petri dish. This thin layer of cells adhering to the glass or plastic dish is overlaid
with a suitable culture medium and is incubated at a suitable temperature.

Aseptic techniques
Bacterial infections, like Mycoplasma and fungal infections, commonly occur in
cell culture creating a problem to identify and eliminate. Thus, all cell culture
work is done in a sterile environment with proper aseptic techniques. Work
should be done in laminar flow with the constant unidirectional flow of HEPA

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filtered air over the work area. All the material, solutions and the whole
atmosphere should be of contamination-free.

Cryopreservation
If a surplus of cells is available from sub-culturing, they should be treated with
the appropriate protective agent (e.g., DMSO or glycerol) and stored at
temperatures below –130°C until they are needed. This stores cell stocks and
prevents original cell from being lost due to unexpected equipment failure or
biological contaminations. It also prevents finite cells from reaching
senescense and minimizes risks of changes in long term cultures.
When thawing the cells, the frozen tube of cells is warmed quickly in warm
water, rinsed with medium and serum and then added into culture containers
once suspended in the appropriate media.

3. Types (especially cell line)

A. Primary cell culture

This is the cell culture obtained straight from the cells of a host tissue. The cells
dissociated from the parental tissue are grown on a suitable container and the
culture thus obtained is called primary cell culture. Such culture comprises
mostly heterogeneous cells and most of the cells divide only for a limited time.
However, these cells are much similar to their parents.
Depending on their origin, primary cells grow either as an adherent monolayer
or in a suspension.

1) Adherent cells
These cells are anchorage dependent and propagate as a monolayer. These
cells need to be attached to a solid or semi-solid substrate for proliferation.
These adhere to the culture vessel with the use of an extracellular matrix
which is generally derived from tissues of organs that are immobile and
embedded in a network of connective tissue. Fibroblasts and epithelial cells
are of such types.
When the bottom of the culture vessel is covered with a continuous layer of
cells, usually one cell in thickness, these are known as monolayer cultures.
Majority of continuous cell lines grow as monolayers. As being single layers,
such cells can be transferred directly to a cover slip to examine under
microscope.
2) Suspension cells

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Suspension cells do not attach to the surface of the culture vessels. These cells
are also called anchorage independent or non-adherent cells which can be
grown floating in the culture medium. Hematopoietic stem cells (derived from
blood, spleen and bone marrow) and tumor cells can be grown in suspension.
These cells grow much faster which do not require the frequent replacement
of the medium and can be easily maintained. These are of homogeneous types
and enzyme treatment is not required for the dissociation of cells; similarly
these cultures have short lag period.
3) Confluent culture and the necessity of sub-culture
After the cells are isolated from the tissue and proliferated under the
appropriate conditions, they occupy all of the available substrate i.e. reach
confluence. For a few days, it can become too crowded for their container and
this can be detrimental to their growth, generally leading to cell death if left
for a long time. The cells thus have to be subculture i.e. a portion of cells is
transferred to a new vessel with fresh growth medium which provides more
space and nutrients for the continual growth of both portions of cells. Hence
subculture keeps cells healthy and in a growing state.
A passage number refers specifically to how many times a cell line has been
sub-cultured. In contrast with the population doubling level in that the specific
number of cells involved is not relevant. It simply gives a general indication of
how old the cells may be for various assays.

B. Secondary cell culture and cell line

When a primary culture is sub-cultured, it is known as secondary culture or cell
line or sub-clone. The process involves removing the growth media and
disassociating the adhered cells (usually enzymatically (.
Sub-culturing of primary cells to different divisions leads to the generation of
cell lines. During the passage, cells with the highest growth capacity
predominate, resulting in a degree of genotypic and phenotypic uniformity in
the population. However, as they are sub-cultured serially, they become
different from the original cell.
On the basis of the life span of culture, the cell lines are categorized into two
types:
Finite cell lines
The cell lines which go through a limited number of cell division having a
limited life span are known as finite cell lines. The cells passage several times
and then lose their ability to proliferate, which is a genetically determined
event known as senescence. Cell lines derived from primary cultures of normal
cells are finite cell lines.

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Continuous cell lines
When a finite cell line undergoes transformation and acquires the ability to
divide indefinitely, it becomes a continuous cell line. Such
transformation/mutation can occur spontaneously or can be chemically or
virally induced or from the establishment of cell cultures from malignant
tissue. Cell cultures prepared in this way can be sub-cultured and grown
indefinitely as permanent cell lines and are immortal.
These cells are less adherent, fast growing, less fastidious in their nutritional
requirements, able to grow up to higher cell density and different in
phenotypes from the original tissue. Such cells grow more in suspension. They
also have a tendency to grow on top of each other in multilayers on culture-
vessel surfaces.

4-Application
A. Vaccines Production
One of the most important uses of cell culture is in research and production of
vaccines. The ability to grow large amounts of virus in cell culture eventually
led to the creation of the polio vaccine, and cells are still used today on a large
scale to produce vaccines for many other diseases, like rabies, chickenpox,
hepatitis B, and measles. In early times, researchers had to use live animals to
grow poliovirus, but due to the development of cell culture techniques, they
were able to achieve much greater control over virus production and on a
much larger scale which eventually develop vaccines and various treatments.
However, continuous cell lines are not used in virus production for human
vaccines as these are derived from malignant tissue or possess malignant
characteristics.

B. Virus cultivation and study

Cell culture is widely used for the propagation of viruses as it is convenient,
economic, easy to handle compared to other animals. It is easy to observe
cytopathic effects and easy to select particular cells on which the virus grow as
well as to study the infectious cycle. Cell lines are convenient for virus research
because cell material is continuously available. Continuous cell lines have been
extremely useful in cultivating many viruses previously difficult or impossible
to grow.

C. Cellular and molecular biology

Cell culture is one of the major tools used in cellular and molecular biology,
providing excellent model systems for studying the normal physiology and
biochemistry of cells (e.g., metabolic studies, aging), the effects of different

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toxic compounds on the cells, and mutagenesis and carcinogenesis. The major
advantage of using cell culture for any of these applications is the consistency
and reproducibility of results that can be obtained from using a batch of clonal
cells.

D. In Cancer Research
Normal cells can be transformed into cancer cells by methods including
radiation, chemicals, and viruses. These cells can then be used to study cancer
more closely and to test potential new treatments.
E. Gene therapy
Cells having a functional gene can be replaced to cells which are having non-
functional gene, and for which the cell culture technique is used.

F. Immunological studies
Cell culture techniques are used to know the working of various immune cells,
cytokines, lymphoid cells, and interaction between disease-causing agents and
the host cells.

G. Others
Cell lines are also used in in-vitro fertilization (IVF) technology, recombinant
protein, and drug selection and improvement.

References
1. Bhatia S et al 2015 Modern Applications of Plant Biotechnology in Pharmaceutical
Sciences
(New York: Academic, Elsevier) pp 164–74
2. Khan F and Tanaka M 2017 Designing smart biomaterials for tissue engineering Int. J.
Mol.
Sci. 19 17
3. Loyola-Vargas VM, Vázquez-Flota FA (2006) An introduction to plant cell culture:
Back to the future.
In: Plant cell culture protocols, Loyola-Vargas VM,
Vázquez-Flota FA (eds) Humana Press, Totowa, New Jersey, pp 1–8
4. Leckie F, Scragg AH, Cliffe KC (1990) The effect of continuous high shear stress on
plant cell suspension cultures.
In: Progress in plant cellular and molecular
biology, Nijkamp HJJ, Van der Plas LHW, Van Aartrijk J (eds) Kluwer Academic
Publishers, The Netherlands, pp 689–693
5. Dracup M (1991) Increasing salt tolerance of plants through cell culture requires
greater understanding of tolerance mechanisms.
Aust J Plant Physiol 18:1–15

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 6. Takeuchi Y, Komamine A (1982) Effects of culture conditions on cell division and
composition of regenerated cell walls in Vinca rosea protoplasts. Plant Cell
Physiol 23:249–255
7. Takeuchi Y, Komamine A (1981) Glucans in the cell walls regenerated from Vinca
rosea protoplasts. Plant Cell Physiol 22:1585–1594
8. Takeuchi Y, Komamine A (1978) Composition of the cell wall formed protoplasts
isolated from cell suspension cultures of Vinca rosea. Planta 140:227–232
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as a pot of gold. Phytochemistry 30:3861–3863
10. Verpoorte R, Van der Heijden R, Memelink J (2000) Engineering the plant cell factory
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11. Shimazaki A, Ashihara H (1982) Adenine and guanine salvage in suspension cultured
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cells of different growth phases and stem tissue of Catharanthus roseus.
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suspension cultures of Agave amaniensis, and its effect on the growth, amino acids
and hecogenin content. Plant Cell Tiss Org Cult 68:287–292
14. Stephan Hellwig, et al. Plant cell cultures for the production of recombinant proteins.
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