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Recombinant DNA Technology - In nature, gene transfers are
rather imprecise, and their range, in tenns of species involved, is
remarkably limited. The above problems are circumvented by the
recombinant DNA technology. A recombinant DNA molecule is produced
by joining together two or more DNA segments usually originating
from different organisms. More specifically, a recombinant DNA
molecule is a vector into which the desired DNA fragment has been
inserted to enable its cloning in an appropriate host. This is
achieved by using specific enzymes for cutting the DNA (restriction
enzymes) into suitable fragments and then for joining together the
appropriate fragments (ligation). In this manner, a gene may be
produced, which contains the. coding region from one organism
joined to regulatory sequences from another organism; such a gene
is called chimaeric gene. Clearly, the capability to produce
recombinant DNA molecules has given man the power and opportunity
to create novel gene functions to suit specific needs. Recombinant
DNA molecules are produced with one of the following three
objectives: (1) to obtain a large number of copies of specific DNA
fragments, (2) to recover large quantities of the protein produced
by the concerned gene, or (3) to integrate the gene in question
into the chromosome of a target organism where it expresses itself.
Even for the latter two objectives, it is essential to first obtain
a large number of copies of the concerned genes. To achieve this,
the DNA segments are integrated into a self-replicating DNA
molecule called vector; most commonly used vectors are either
bacterial plasmids or DNA viruses. All these steps concerned with
piecing together DNA segments of diverse origin and placing them
into a suitable vector together constitute recombinant DNA
technology. The DNA segment to be cloned is called DNA insert.
Recombinant DNAs are introduced into a suitable organism, usually a
bacterium; this organism is called host, while the process is
called transformation. The transformed host cells are selected and
cloned. The recombinant DNA present in such clones would replicate
either in synchrony with or independent of the host cell; the gene
present in 'the vector mayor may not express itself, i.e., direct
the synthesis of concerned polypeptide. The step concerned with
transformation of a
suitable host with recombinant DNA, and cloning of the
transformed cells is called DNA cloning or gene cloning. However,
often DNA or gene cloning is taken to include both the development
of recombinant DNAs as well as their cloning in a suitable host.
Similarly, often the term recombinant DNA technology is used as a
synonym for DNA or gene cloning used in the broader sense. A rather
popular term for these activities is genetic engineering. A clone
consists of asexual progeny of a single individual or cell, while
the process/technique of producing a clone is called cloning. As a
result, all the individuals of a clone have the same genotype,
which is also identical with that of the individual from which the
clone was derived. Therefore, the genomes present in members of a
single clone are also identical; this applies to the recombinant
DNA as well. Therefore, gene or DNA cloning produces large numbers
of copies of the gene/DNA being cloned.
Restriction Endonucleases - Endonucleases are enzymes that
produce internal cuts, called cleavage, in DNA molecules. Many
endonucleases cleave DNA molecules at random sites. But a class of
endonucleases cleaves DNA only within or near those sites, which
have specific base sequences; such endonucleases are known as
restriction endonucleases, and the sites recognised by, them are
called recognition sequences or recognition sites. The recognition
sequences are different and specific for the different restriction
endonucleases or restriction enzymes.Restriction enzymes were
discovered due to and named after the phenomenon of host
restriction of bacterial phages. The presence bf restriction
enzymes was postulated by W. Arber during 1960s, while the first
true restriction endonuclease was isolated in 1970. Smith, Nathans
and Arber were awarded the Nobel Prize for Physiology and Medicine
in 1978 for the discovery of endonucleases. Restriction
endonucleases are indispensable for DNA cloning and sequencing.
They serve as the tools for cutting DNA molecules at predetermined
sites, which is the basic requirement for gene cloning or
recombinant DNA technology.
Restriction modification systemFrom Wikipedia, the free
encyclopedia
The restriction modification system (RM system) is used by
bacteria, and perhaps other prokaryotic organisms to protect
themselves from foreign DNA, such as the one borne by
bacteriophages. This phenomenon was first noticed in the 1950s.
Certain bacteria strains were found to inhibit (restrict) the
growth of viruses grown in previous strains. This effect was
attributed to sequence-specific restriction enzymes.
Bacteria have restriction enzymes, also called restriction
endonucleases, which cleave double stranded DNA at specific points
into fragments, which are then degraded further by other
endonucleases. This prevents infection by effectively destroying
the foreign DNA introduced by an infectious agent (such as a
bacteriophage). Approximately one quarter of known bacteria possess
RM systems and of those about one half have more than one type of
system. Restriction enzymes only cleave at specific sequences of
DNA which are usually 4-6 base pairs long, and often palindromic.
Given that the sequences that the restriction enzymes recognize are
very short, the bacterium itself will almost certainly have many of
these sequences present in its own DNA. Therefore, in order to
prevent destruction of its own DNA by the restriction enzymes, the
bacterium marks its own DNA by adding methyl groups to it. This
modification must not interfere with the DNA base-pairing, and
therefore, usually only a few specific bases are modified on each
strand.
Contents[hide]
1 Types of restriction modification system 2 Uses 3 See also 4
References
[edit] Types of restriction modification systemThere are Four
kinds of restriction modification system: type I, type II, type
IIS, type III and Type IV, all with restriction enzyme activity and
a methylase activity. They were named in the order of discovery,
although the type II system is the most common. Type I systems are
the most complex, consisting of three polypeptides: R
(restriction), M (modification), and S (specificity). The resulting
complex can both cleave and methylate DNA. Both reactions require
ATP, and cleavage often occurs a considerable distance from the
recognition site. The S subunit determines the specificity of both
restriction and methylation. Cleavage occurs at variable distances
from the recognition sequence, so discrete bands are not easily
visualized by gel electrophoresis. Type II systems are the simplest
and the most prevalent. Instead of working as a complex, the
methyltransferase and endonuclease are encoded as two separate
proteins and act independently (there is no specificity protein).
Both proteins recognize the same recognition site, and therefore
compete for activity. The methyltransferase acts as a monomer,
methylating the duplex one strand at a time. The endonuclease acts
as a homodimer, which facilitates the cleavage of both strands.
Cleavage occurs at a defined position close to or within the
recognition sequence, thus producing discrete fragments during gel
electrophoresis. For this reason, Type II systems are used in labs
for DNA analysis and gene cloning.
Type III systems have R and M proteins that form a complex of
modification and cleavage. The M protein, however, can methylate on
its own. Methylation also only occurs on one strand of the DNA
unlike most other known mechanisms. The heterodimer formed by the R
and M proteins competes with itself by modifying and restricting
the same reaction. This results in incomplete digestion.[1][2]
[edit] UsesRM systems can be cloned into plasmids and selected
because of the resistance provided by the methylation enzyme. Once
the plasmid begins to replicate, the methylation enzyme will be
produced and methylate the plasmid DNA, protecting it from a
specific restriction enzyme. Some viruses have evolved ways of
subverting the restriction modification system, usually by
modifying their own DNA, by adding methyl or glycosyl groups to it,
thus blocking the restriction enzymes. Other viruses, such as
bacteriophages T3 and T7, encode proteins that inhibit the
restriction enzymes. To counteract these viruses, some bacteria
have evolved restriction systems which only recognize and cleave
modified DNA, but do not act upon the host's unmodified DNA. Some
prokaryotes have developed multiple types of restriction
modification systems.
Types of Restriction Endonucleases - There are three distinct
types of restriction endonucleases. Type I restriction
endonucleases are complex endonucleases, and have recognition
sequences of about 15 bp; they cleave the DNA about 1000 bp away
from the 5'-end of the sequence "TCA" located within the
recognition site, e.g., EcoK, EcoB etc. Type II restriction
endonucleases are remarkably stable and induce cleavage either, in
most cases, within their recognition sequences or very close to
them. More than 350 different type II endonucleases with over 100
different recognition sequences are known. They require Mg2+ ions
for cleavage. The first type II enzyme to be isolated was Hind II
in 1970. Only type II restriction endonucleases are used for
restriction mapping and gene cloning. Type III restriction
endonucleases are intermediate between the type I and type II
enzymes; they cleave DNA in the immediate vicinity of their
recognition sites, e.g., EcoPl, EcoP15, Hind III etc. Nomenclature
of Restriction Endonucleases - The nomenclature of restriction
endonucleases follows a general pattern. (1) The first letter of
the name of genus in which a given enzyme is first discovered is
written in capital. (2) This is followed by the first two letters
of species name of the organism. These three letters
are generally written in italics, e.g., Eco from Escherichia
coli, Hin from Haemophilus influenzae, etc. (3) Strain or type
identification is depicted next in Roman. e.g., Ecok; (4) When an
organism produces more than one enzyme, they are identified by
sequential Roman numerals, e.g., the different enzymes produced by
H. influenzae strain Rd are named Hind II, Hind III, etc. ome
Restriction Endonucleases Restriction endonuclease AvaI* AluI BamHI
EcoRI EcoRII** BglII HindII HindIII HindII HpaI HpaII HinfI* NIaIII
PstI Sau3A TaqI Source (organism an strain) Anabaena variabilis (A
TCC 27892) Arthrobacter luteus Bacillus amyloliquefaciens H
Escherichia coli Ry13 E. coli R245 Bacillus globigli Haemophilus
influenzae Rd H. influenzae Rd H. influenzae PI H. parainfluenzae
H. parainfluenzae H. influenzae Rf Neisseria lactamica Providencia
stuartii Staphylococcus aureus 3A Thermus aquaticus YTI Recognition
sequence C/Py CG Pu G G Pu GC Py/C AG/CT TC/GA G/G AT CC C C TA G/G
G/AA TT C C TT AA/G /CCA (T) GG GGT(A)CC/ A/G ATCT TC TAG/A GT
Py/Pu AC CA Pu/Py TG A/A GC T T T T CG A/A G/G C C C C G/G GTT/ AAC
CAA/TTG C/C GG GG C/C G/A NT C C T NA/G CATG/ /GTAC C T GCA/G G/A
CGTC /GATC CT AG/ T/C GA AG C/T
* Pu, either of the two purines (adenine or guanine) Py, either
of the two pyrimidines (thymine or cytosine) N, any of the four
bases (adenine, guanine, thymine or cytosine) ** The base given
within parenthesis, e.g., (T) may occur in place of the preceding
base, e.g., A. Recognition Sequences For Type II Endonucleases -
The recognition sequences for Type II endonucleases form
palindromes with rotational symmetry. In a palindrome, the base
sequence in the second half of a DNA strand is the mirror image of
the sequence in its first half; consequently, the complementary DNA
strand of a double helix also shows the same situation. But in a
palindrome with rotational symmetry, the base sequence in the first
half of one strand of a DNA double helix is the mirror image of the
second half of its complementary strand. Thus in such palindromes,
the base sequence in both the strands of a DNA duplex reads the
same when read from the same end (either 5' or 3') of both the
strands. Most of the type II restriction endonucleases have
recognition sites of 4, 5 or 6 bp (base pairs), which are
predominantly GC-rich. Longer palindromic target sequences are also
known, and so are nonpalindromic ones (specific for some enzymes).
Some restriction enzymes have ambiguities in their recognition
sites, e.g. EcoRII, so that they may recognise upto 4 different
target sequences. Cleavage Pattern of Type II Restriction
Endonucleases - Most type II restriction endonucleases cleave the
DNA molecules within their specific recognition sequences, but some
produce cuts immediately outside the target sequence, e.g., NlaIII,
Sau3A, etc. These cuts are either (1) staggered or (2) even,
depending on the enzyme. Most enzymes produce staggered cuts in
which the two strands of a DNA double helix are cleaved at
different locations; this generates protruding (3'- or 5'-) ends,
i.e., one strand of the double helix extends some bases beyond the
other: Due to the palindromic (symmetrical) nature of the target
sites, the two protruding ends generated by such a cleavage by a
given enzyme have complementary base sequence. As a result, they
readily pair with each other; such ends are called cohesive or
sticky ends. An important consequence of this fact is that when
fragments generated by a single restriction enzyme from different
DNAs are mixed, they join together due to their sticky ends.
Therefore, this property of the restriction enzymes is of great
value for the construction of recombinant DNAs. Some restriction
enzymes, on the other hand, cut both the strands of a DNA molecule
at the same site so that the resulting termini or ends have blunt
or flush ends in which the two strands end at the same point. The
blunt cut ends also can be effectively utilized for construction of
recombinant DNAs following one of several strategies.
Modification of Cut Ends - The 3'-ends of DNA strands always
carry a free hydroxyl (-OH) group, while their 5'-ends always bear
a phosphate group. Often the ends produced by restriction enzymes
have to be modified for further manipulation of the fragments; some
of the modifications are summarised below. 1. Removal of the
5'-phosphate group of vector DNA by alkaline phosphatase treatment
in order to prevent vector circularization during DNA insert
integration. 2. Addition of a phosphate group to a free 5'-hydroxyl
group by T4 polynucleotide kinase. 3. Removal of the protruding
ends by digestion with, say, S1 nuclease; this enzyme digests both
3'- and 5'-protruding ends. 4. Filling in of the protruding ends by
extending the recessed (shorter) strand with, say, Klenow fragment
of E. coli DNA polymerase I. (Both the strategies 3 and 4 generate
blunt ends which can be ligated by T4 polynucleotide ligase.) 5.
Synthesis of single-stranded tails (protruding ends) at the 3'-ends
of blunt ended fragments by the enzyme terminal deoxynucleotidyl
transferase; this is called tailing. This reaction can be used to
generate protruding ends of defined sequence, e.g., poly-A tails on
the 3'ends of the DNA insert and poly- T tails on the 3'-ends of
the vector; the protruding ends of the DNA insert and the vector
will, therefore, base pair under annealing conditions. 6. Linker
and/or adaptor molecules can be joined to the cut ends. Linkers are
short, chemically synthesized, self complementary, double stranded
oligonucleotides, which contain within them one or more restriction
endonuclease sites, e.g., linker 5' -CCGAA TTCGG (only one strand
of the linker is shown here) contains one EcoRI site. Linkers are
joined with blunt ended DNA fragments; cleavage of the linker with
the appropriate restriction enzyme creates suitable cohesive
protruding ends Linkers create cohesive ends Oft blunt ended DNA
fragments, and on fragments having unmatched or undefined sequences
in their protruding ends. In the latter situation, the DNA
fragments are first made blunt-ended, following which the selected
linkers are ligated to them by T 4 ligase. 7. Adaptors are short,
chemically synthesized- DNA double strands, which can be used to
link the ends of two DNA molecules that have different sequences at
their ends. There are different kinds of adaptors suited for
different purposes. For example, a conversion adaptor is used to
join a DNA fragment or insert cut with one restriction enzyme, say,
EcoRI, with a vector opened with another enzyme, e.g., BamHI. These
adaptors have the recognition sequences of different endonucleases
at their ends. For example, the conversion adaptor has recognition
sequence for BamHI at one end and that for EcoRI at the other. This
adaptor can be used to convert the cohesive end generated by BamH1
to one produced by EcoRI or vice versa.
Steps in Gene Cloning - The entire procedure of cloning or
recombinant DNA technology may be classified into the following
five steps for the convenience in description and on the basis of
the chief activity performed. 1. Identification and isolation of
the desired gene or DNA fragment to be cloned. 2. Insertion of the
isolated gene in a suitable vector. 3. Introduction of this vector
into a suitable organism/cell called host (transformation). 4.
Selection of the transformed host cells. 5.
Multiplication/expression/integration followed by expression of the
introduced gene in the host.
Selection of Recombinant Clones - When recombinant DNA is
constructed and used for transformation of E. coli, cells following
types of bacterial cells are obtained: (1) majority of the cells
are nontransformed, (2) a proportion of the transformed cells
contain unaltered vector, while (3) the remainder cells have
recombinant DNA. The first objective of cloning experiments is to
identify and isolate those small number of cells that contain the
recombinant DNA from among a very large number of nontransformed
cells. Since the DNA inserts are generally mixtures, particularly
when cDNA preparations and genomic DNA fragments are used, the
various transformed clones would contain a variety of different DNA
inserts. The next step, therefore, is to identify the clone having
the desired DNA insert from among the large number of clones
containing the recombinant DNAs. Suitable selection strategies have
been devised to achieve these two critical objectives; this is the
most important step in DNA cloning. Identification of Clones Having
Recombinant DNAs - The second step consists of identification and
isolation of those clones that are transformed by the recombinant
DNAs from among those that contain the unaltered vector. This may
be achieved in one of several ways listed below. 1. In case the
vector has two selectable markers, e.g., pBR322, the DNA insert may
be placed within one of these markers, say, ampT gene. The other
marker, in this case, tetr, is used for
elimination of the nontransformed cells. The transformed clones
are then replicaplated on ampicillin containing medium. The clones
containing the recombinant DNAs will be sensitive to ampicillin due
to inactivation of the gene ampT by insertion of the DNA fragment.
Such clones are identified and isolated from the master plate. 2.
Some vectors contain a gene, or sometimes only part of a gene,
which complements a function missing in their host cells, e.g.,
gene lacZ in the pUC vectors, which complements such lacZ- E. coli
strains in which lacZ is deleted. The same combination is used for
some A. vectors and M13 phage vectors. In all such cases, the DNA
insert is so placed that it disrupts the expression of lacZ.
Therefore, E. coli cells containing the recombinant DNA are
deficient in -galactosidase and produce white colonies or plaques
on a medium containing X-gal and IPTG. On the other hand, tells
having the unchanged vector produce active -galactosidase and give
rise to blue colonies or plaques on the same medium. This allows an
easy identification of the clones containing the recombinant DNAs.
3. When the DNA insert codes for a gene product, which is defective
in the auxotrophic host cells, a direct selection for the
recombinant DNA is possible. The host cells are grown on a medium
lacking the compound needed by the auxotrophic host; only those
cells, which contain the recombinant DNA can grow and form
colonies. Obviously, this approach is limited in application. 4.
Similarly, selection by suppression of nonsense mutations present
in the host also permits a direct selection for the recombinant
DNA. 5. Some A. vectors retain the lysogenic function as well,
e.g., gt10. In such vectors, the DNA insert may be placed within
the lysis repressor gene cI- so that the vector becomes cI. As a
result, cells transfected by the recombinant DNA will give rise to
clear plaques, whereas those infected by the unaltered vector will
yield cloudy or turbid plaques. Thus the recombinant DNAs are
readily identified and isolated. 6. Some vectors, e.g., A.
replacement vectors and cosmids, are much shorter than the minimum
genome length needed for their packaging within virus particles. In
such cases, the length of DNA insert can be so adjusted as to allow
the packaging of only the recombinant DNA. This provides an
efficient selection strategy for recombinant DNA. Selection of
Clone Containing A Specific DNA Insert -Once we obtain a population
of recombinant clones the next step is to identify a clone, which
has the DNA insert of interest. The technique used for
identification has to be highly precise and extremely sensitive to
allow an accurate detection of a single clone from among the
thousands obtained from a cloning experiment. The various
strategies used for the purpose are briefly outlined below.
Colony Hybridization. The most efficient and rapid strategy for
identification of a clone having the desired insert uses the
technique of colony hybridization. The bacterial colonies are
replicaplated or phage plaques are directly lifted on
nitrocellulose filters, the cells are lysed and their DNA is
denatured, the filter is incubated with the specific radioactive
32p-labelled) probe under anealing conditions. After some time, the
probe is washed out leaving only those probe molecules that have
hybridized with the denatured DNA from bacterial cells or phage
particles. The colonies/plaques with whose DNA the probe has
hybridized are identified by autoradiography; these contain the
desired DNA insert. These colonies/plaques are isolated from the
master plate used for replica plating.A very large number of
colonies or plaques (upto 10,000 plaques) can be lifted on to a
single 10 cm diameter filter. But it is essential that a specific
probe for the DNA insert is available. A probe is a polynucleotide
(DNA or RNA; usually small molecules of as few as 15 bases, but
more often of 2530 bases) molecule of a specific base sequence,
which is used to detect DNA molecules having the same base sequence
by complementary base pairing. Generally, the probes are labelled
with 32p to enable autoradiography for an easy identification of
the DNA samples that base-pair with the probe. It is desirable that
the probes are single-stranded to avoid pairing between the two
strands of the probe itself. Either DNA or RNA can be used as
probe. There are several approaches for developing specific probes.
Other Approaches. When specific probes are not available, many
indirect approaches may be used for the identification of clones
having the desired DNA insert. These procedures are not generally
convenient for screening of a large number of clones. Two of such
procedures, called (1) hybrid arrested translation (HART) and (2)
hybrid selection, use in vitro translation systems and then
identification of the resulting polypeptide(s).It is, therefore,
necessary that the protein product of the DNA insert being searched
should be known, at least in terms of its electrophoretic mobility.
Complementation. The cloned DNA insert may express itself in the
bacterial cells; this is possible for prokaryotic genes, some yeast
genes and for eukaryotic cDNAs cloned in suitable expression
vectors. Eukaryotic sequences isolated from genomic DNA have to be
expressed in appropriate eukaryotic hosts, e.g., yeast cells,
animal cells m culture, etc. If the protein produced by the desired
DNA insert is deficient in the
host cells, this insert will correct the deficiency of the cells
transformed by it, i.e., will complement the deficiency of host
cells. This can be stated in general terms as follows. The host
cells are deficient in a protein A, i.e., they are A-. These cells
can be used to isolate the DNA fragment coding for protein A from a
mixture of DNA fragments. Expression of recombinant DNAs are
prepared from the DNA fragments and A- host cells are transformed;
these cells are now cultured under selective conditions that
require functional A product. Only those host cells that contain
the DNA insert encoding protein A will be able to multiply under
the selective conditions (since the DNA insert will provide
functional protein A). This strategy is limited in
application by the availability of appropriate host cells.
Unique Gene Products. Alternatively, the protein product of DNA
insert can be identified by its unique function, i.e., a function
not performed by the proteins of nontransformed host cells. Such
functions may relate to enzyme activities or hormone effects for
which appropriate assays exist. Antibodies Specific to the Protein
Product. Finally, if the protein lacks a recognizable and
measurable function, it can be detected by using specific
antibodies. A practical approach is to divide the large number of
recombinant clones into a convenient number of groups and to assay
for the presence of the protein. The positive group is again
divided into subgroups and assayed. In this manner, the positive
groups are subdivided again and again till a single positive clone
is identified. This approach is applicable to the previous strategy
as well. The identification of proteins using antibodies may be
achieved by western blotting, precipitation and electrophoresis or
ELISA (enzyme-linked immunosorbent assay; Appendix-2.IX).
Colony/Plaque Screening with Antibodies. An efficient and rapid
screening using antibodies is as follows. The antibody specific to
the concerned gene product (i.e., protein) is spread uniformly over
a solid support, e.g., plastic or paper disc, which is placed in
contact with an agar layer containing lysed bacterial colonies or
phage plaques. If any clone is producing the protein in question,
it will bind to the antibody molecules present on the disc. The
disc is removed from the agar, is treated with a second
radiolabelled (generally with L25I) antibody, which is also
specific to the same protein but in a region different from that
recognised by the first antibody. These antibodies, therefore, will
also bind to the protein moleculed held by the first antibody; the
location of radioactivity on the disc is determined by
autoradiography. The colonies/plaques producing the protein are
then identified and isolated from the master plate. This technique
is analogous to colony hybridization and is able to screen large
numbers of clones rather rapidly. But for this technique we require
two different antibodies, which bind to two distinct domains of the
desired protein, and this protein must not be produced by the
nontransformed host cells. FACS. In case of animal cells, an
automated system, called fluorescence activated cell sorter (FACS),
can be used for very rapid (upto 1,000 cells/sec) sorting of
transformed cells. This is applicable to all the genes whose
products become arranged on the cell surface and are available for
binding of specific antibodies. Therefore, these proteins must not
be produced by the nontransformed host cells. The antibody
molecules are attached to a fluorescent molecule and the
transformed cells are treated with this antibody specific for the
desired protein. The cells containing on their surface the protein
in
question will interact with the fluorescent antibodies. Cells
are then passed one by one in a stream between a laser and a
fluorescence detector. The cells which fluoresce are deflected into
a microculture tray, while the nonfluorescing cells are drawn away
by an aspirator. This approach is also applicable to the genes
encoding receptor proteins present on the cell surface; in such
cases, fluorescent ligands (the concerned molecule to which the
receptor binds) are used in the place of fluorescent
antibodies.
Cell culturegrowth factors which promotes cell proliferation,
cell attachment and adhesion factors. Serum is obtained from human
adult blood, placental, cord blood, horse blood, calf blood. The
other forms of biological fluids used are coconut water, amniotic
fluid, pleural fluid, insect haemolymph serum, culture filtrate,
aqueous humour, from eyes etc.
iii) Tissue extracts for example Embryo extracts- Extracts from
tissues such as embryo, liver, spleen, leukocytes, tumour, bone
marrow etc are also used for culture of animal cells . Synthetic
media Syntheic media are prepared artificially by adding several
organic and inorganic nutrients, vitamins, salts, serum proteins,
carbohydrates, cofactors etc. Different types of synthetic media
can be prepared for a variety of cells and tissues to be cultured.
Synthetic media are of two types- Serum containing media (media
containing serum) and serum- free media (media with out serum).
Examples of some media are: minimal essential medium (MEM), RPMI
1640 medium, CMRL 1066, F12 etc. Advantages of serum in culture
medium are: i) serum binds and neutralizes toxins, (ii) serum
contains a complete set of essential growth factors, hormones,
attachment and spreading factors, binding and transport proteins,
(iii) it contains the protease inhibitors, (iv) it increases the
buffering capacity, (v) it provides trace elements. Disadvantages
of serum in culture medium are: (i) it is not chemically defined
and therefore its composition varies a lot, (ii) it is sometimes
source of contamination by viruses, mycoplasma, prions etc, (iii)
it increases the difficulties and cost of down stream processing,
(iv) it is the most expensive component of the culture medium. 4)
pH- Most media maintain the pH between 7 and 7.4. A pH below 6.8
inhibits cell growth. The optimum pH is essential to maintain the
proper ion balance, optimal functioning of cellular enzymes and
binding of hormones and growth factors to cell surface receptors in
the cell cultures. The regulation of pH is done using a variety of
buffering systems. Most media use a bicarbonate-CO2 system as its
major component.
5) Osmolality- A change in osmolality can affect cell growth and
function. Salt, Glucose and Amino acids in the growth media
determine the osmolality of the medium. All commercial media are
formulated in such a way that their final osmolality is around 300
mOsm. CELL BASED THERAPY The animal cell culture techniques are
used in replacing the damaged and dead cells with normal and
healthy cells using the stem cell technology. This therapy is
called Cell-Based therapy which involves the use of stem cell
technology involving the replacement of damaged and dead cells with
normal and healthy cells. This is used to treat blood cancer, and
other neuro-degenerative diseases etc. APPLICATIONS OF ANIMAL CELL
CULTURE The animal cell cultures are used for a diverse range of
research and development. These areas are: a) production of
antiviral vaccines, which requires the standardization of cell
lines for the multiplication and assay of viruses. b) Cancer
research, which requires the study of uncontrolled cell division in
cultures.
c) Cell fusion techniques.
d) Genetic manipulation, which is easy to carry out in cells or
organ cultures.
e) Production of monoclonal antibodies requires cell lines in
culture.
f) Production of pharmaceutical drugs using cell lines.
g) Chromosome analysis of cells derived from womb.
h) Study of the effects of toxins and pollutants using cell
lines.
i) Use of artificial skin.
j) Study the function of the nerve cells. Somatic Cell Fusion
One of the applications of animal cell culture is the production of
hybrid cells by the fusion of different cell types. These hybrid
cells are used for a the following purposes: (i) study of the
control of gene expression and differentiation, (ii) study of the
problem of malignancy, (iii) viral application,
(iv) gene mapping, (v) production of hybridomas for antibody
production. In 1960s, in France for the first time, the hybrid
cells were successfully produced from mixed cultures of two
different cell lines of mouse. Cells growing in culture are induced
by some of the viruses such as Sendai virus to fuse and form
hybrids. This virus induces two different cells first to form
heterokaryons. During mitosis, chromosomes of heterokaryon move
towards
the two poles and later on fuse to form hybrids. It is important
to remove the surface carbohydrates to bring about cell fusion.
Some other chemicals like polyethylene glycol also induce somatic
cell fusion. Many commercial proteins have been produced by animal
cell culture and there medical application is being evaluated. FIG
SHOWING THE PRODUCTION OF T-PA Tissue Plasminogen activator (t-PA)
was the first drug that was produced by the mammalian cell culture
by using rDNA technology. The recombinant t-PA is safe and
effective for dissolving blood clots in patients with heart
diseases and thrombotic disorders.
When Monoclonal antibodies are used as enzymes using the
technique of enzyme engineering, then they are calledabz y mes.
Using animal cell cultures, it is also possible to produce
Polyclonal Antibodies. Polyclonal antisera are derived from many
cells therefore contains heterogeneous antibodies that are specific
for several epitopes or an antigen. SCALE-UP OF ANIMAL CELL
CULTURE
Modifying a laboratory procedure, so that it can be used on an
industrial scale is called scaling up. Laboratory procedures are
normally scaled up via intermediate models of increasing size. The
larger the plant, the greater the running costs, as skilled people
are required to monitor and maintain the machinery.The first
prerequisite for any large scale cell culture system and its
scaling up is the establishment of a cell bank. Master cell banks
(MCB) are first established and they are used to develop Master
Working Cell Banks (MWCB). The MWCB should be sufficient to feed
the production system at a particular scale for the predicted life
of the product. The cell stability is an important criteria so MWCB
needs to be repeatedly subcultured and each generation should be
checked for changes. A close attention should be paid to the volume
of cultured cells as the volume should be large enough to produce a
product in amounts which is economically viable. The volume is
maintained by a) increasing the culture volume, (b) by increasing
the concentration of cells in a reactor by continuous perfusion of
fresh medium, so that the cells keep on increasing in number
without the dilution of the medium. A fully automated bioreactor
maintains the physicochemical and biological factors to optimum
level and maintains the cells in suspension medium. The most
suitable bioreactor used is a
compact-loop bioreactor consisting of marine impellers. The
animal cells unlike bacterial cells, grow very slowly. The main
carbon and energy sources are glucose and glutamine. Lactate and
ammonia are their metabolic products that affect growth and
productivity of cells. So, the on-line monitoring of glucose,
glutamate, and ammonia is carried out by on line flow injection
analysis (FIA) using gas chromatography (GC), high performance
liquid chromatography (HPLC) etc. In batch cultures, mainly Roller
Bottles with Micro Carrier Beads (for adherent cells) and spinner
flasks (for suspension cultures) are used in Scale-up of animal
cell culture process. Roller Bottles The Roller bottles provide
total curved surface area of the micro carrier beads for growth.
The continuous rotation of the bottles in the CO2 incubators helps
to provide medium to the entire cell monolayer in culture.The
roller bottles are well attached inside a specialized CO2
incubators. The attachments rotate the bottles along the long axis
which helps to expose the entire cell monolayer to the medium
during the one full rotation. This system has the advantage over
the static monolayer culture: (a) it provides increase in the
surface area, (b)
provides constant gentle agitation of the medium, (c) provides
increased ratio of surface area of medium to its volume, which
allows gas exchange at an increased rate through the thin film of
the medium over the cells. Typically, a surface area of 750-1500
cm2 with 200500 ml medium will yield 1-2x108cells.
DIAGRAM SHOWING THE ROLLER BOTTLE CELL CULTURE
Micro Carrier Beads Micro carrier beads are small spherical
particles with diameter 90-300 micrometers, made up of dextran or
glass. Micro Carrier beads, increase the number of adherent cells
per flask. These dextran or glass-based beads come in a range of
densities and sizes. The cells grow at a very high density which
rapidly exhausts the medium and therefore the medium has to be
replaced for the optimum cell growth. At the recommended
concentration when the microcarriers are suspended they provide
0.24 m2 area for every 100 ml of culture flask. Spinner cultures
The spinner flask, was originally developed to provide the gentle
stirring of microcarriers but are now used for scaling up the
production of suspension cells. The flat surface glass flask is
fitted with a Teflon paddle that continuously turns and agitates
the medium. This stirring of the medium improves gas exchange in
the cells in culture. The spinner flask used at commercial scale
consists of one or more side arms for taking out samples and
decantation as well. TYPES OF CELL 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 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.
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)Kar yo ty pi ng - 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
The culture animal material is washed in balanced salt solution
to avoid contamination. The tissue to be cultured should be
properly sterilized with 70% ethanol and removed surgically under
aseptic conditions.
Disaggregation of tissue To obtain the cell suspension for
primary cell culture, the tissue is disintegrated either
mechanically or by using enzymes. (i) Physical or mechanical
disaggregation- After removing the tissue under aseptic conditions,
it is pressed through a sieve of 100 micrometer. It is then kept in
a sterile Petri dish containing buffered medium with balanced salt
solution. The cells are then alternately passed through the sieve
of decreasing pore size (50 micrometer and 20 micrometer mesh). The
debris which remains on the sieve is discarded and the medium
containing cells is collected and cells are counted by using
haemocytometer. This method is cheap and quick but it damages a lot
of cells. (ii) Enzymatic disaggregation- In this method, enzymes
are used for dislodging the cells of tissues. The two important
enzymes used in tissue disaggregation are-collagenase and trypsin.
-a) Collagenase- The intracellular matrix contains collagen
therefore collagenase is used for disaggregation of embryonic,
normal as well as malignant tissues. The tissues are kept in medium
containing antibiotics and then dissected into pieces in basal salt
solution. After washing the chopped tissue with distilled water, it
is transferred to complete medium containing collagenase. After a
few days (around 5 days), the mixture is pipetted so
that the medium gets dispersed. The whole treatment is left for
sometimes during which the epithelial cells settle on bottom of
test tubes. The enzyme collagenase is removed by centrifugation.
Suspension consists of cells which are then plated out on the
medium. (b) TrypsinUse of trypsin for disaggregation is called
trypsinization. On the basis of role of temperature on trypsin, the
activity of trypsin is of two types- Cold trypsinization and warm
trypsinization. Cold trypsinization- The sample tissue to be
disaggregated is chopped into 2-3 small pieces and kept in sterile
glass vial. The tissues are subsequently washed with sterile water
and dissected and then kept in BSS. The whole content is then
placed on ice and soaked in cold trypsin for 4-6 hours to allow the
penetration of enzymes in tissue. After this the trypsin is removed
and the tissue is incubated at 36.50C for 20-30 minutes. About 10
ml of medium containing serum is added to the vials containing the
cells and the cells are dispersed by repeated pipetting. The cells
are counted by haemocytometer and are plated and incubated for
48-72 hours for cell growth.
Warm trypsinization- The initial steps are the same as in cold
trypsinization however, in this case the tissue pieces are treated
with warm trypsin (36.50C). The tissues are stirred for 4 hours and
then pieces are allowed to settle down. The disassociated cells are
collected at every 30 minutes. The process is repeated by adding
fresh trypsin back to pieces and incubating the contents. The
trypsin is removed by centrifugation after 3-4 hours during which
the complete disaggregation of tissues takes place. The glass vials
containing dispersed cells are then placed on ice. The cells are
counted using haemocytometer and cell density is maintained at an
appropriate number. The cells are then plated on medium and
incubated for 48-72 hours for cell growth. (iii) Treatment with
chelating agents- The tissues like epithelium (which needs Ca2+ and
Mg2+ ions for its integrity are treated with chelating agents such
as citrate and ethylene- diamine-tetra-acetic acid (EDTA).
Chelating agents are mainly used for production of cell suspensions
from established cultures of epithelial type. STEM CELL TECHNOLOGY
Stem cells retain the capacity to self renew as well as to produce
progeny with a restricted mitotic potential and restricted range of
distinct types of differentiated cell they give rise to. The
formation of blood cells also called haematopoiesis is the
classical example of
concept of stem cells. Indirect assay methods were developed to
identify the haematopoietic stem cells. The process ofhaematopoeis
is occurs in the spleen and bone marrow in mouse. In human beings
about 100,000 haematopoietic stem cells produce one billion RBC,
one billion platelets, one million T-cells, one million B cells per
kg body weight per day. Several methods have been developed to
study haematopoiesis and stem cells: a) Repopulation assay- Edmens
Snells group created mice which were genetically identical by
mating of sibling mice after 21 generations. Two groups of mice
were lethally X- irradiated to destroy their blood cell forming
capacity. One of this group was injected with marrow cells from the
femur bone of a normal and healthy albino mice. It was observed
that this group survived whereas the mice in the other group died.
The spleen of mice which survived had the colonies of the bone
marrow cells just like bacterial colonies on a Petri plate. This
came to be known as colony forming units of spleen (CFU-S) and the
technique is known as repopulation assay. b) The in vitro clonal
assay- In this assay, the stem cells proliferate to form colonies
of
differentiated cells on semi-solid media. This assay helps in
identifying growth factors required for the formation of blood
cells from the primitive stem cells. One of the first
commercialized
Genetic Engineering of animal cells and their applications The
mammalian cells are genetically modified by introducing the genes
needed for specific purposes such as production of specific
proteins or to improve the characteristics of a cell line. The
methods used to introduce the foreign genes/DNA into mammalian
cells are: Electroporation, Lipofection, Microinjection and/or
fusion of mammalian cells
with bacteria or viruses. After the integration of the foreign
DNA into the mammalian cells, the transfected/transformed cells are
selected by using suitable markers. Some of such markers in use
are: Viral thymidine kinase, Bacterial dihydrofolate reductase,
Bacterial neomycin phosphotransferase. It has been possible to
overproduce several proteins in mammalian cells through genetic
manipulations e.g. tissue plasminogen activator, erythropoietin,
interleukin-2, interferon- beta, clotting factors VIII and IX,
tumor necrosis factors. The recombinant mammalian cells are also
conveniently used for the production of monoclonal antibodies.
Manipulation of Gene Expression in Eukaryotes The eukaryotic
organisms have the capability to bring about the post-translational
modifications such as glycosylation, phosphorylation, proteolytic
cleavage etc which ultimately helps in the production of stable and
biologically active proteins. Due to these reasons the use of
eukaryotic expression system is preferred however it is difficult
to conduct experiments with eukaryotic cells. The introduction of a
foreign DNA into animal cells is called transfection. The insert
DNA in the eukaryotic cells may be associated with vector or
integrated into the host
chromosomal DNA. Among the various hosts used for the expression
of cloned genes, the common yeast Saccharomyces cerevisiae is the
most extensively used. Besides this, the cultured insect cells are
in use for expressing cloned DNAs. Baculoviruses exclusively infect
insect cells. The DNA of these viruses encode for several products
and their productivity in cells is very high to the extent of more
than 10,000 times compared to mammalian cells. The baculoviruses
not only carry a large number of foreign genes but can also express
and process the products formed. By using baculovirus as an
expression vector system, a good number of mammalian and viral
proteins have been synthesized. The most commonly used baculovirus
is Autographa californica multiple nuclear polyhedrosis virus
(AcMNPV). It grows on the insect cell lines and produce high levels
of polyhedrin or a recombinant protein. The mammalian cell
expression vectors are used for the production of specific
recombinant proteins and to study the function and regulation of
mammalian genes. However, large-scale production of recombinant
proteins with engineered mammalian cells is costly. The mammalian
vector contains a eukaryotic origin of replication from an animal
virus such as
Simian virus 40 (SV 40) and a prokaryotic origin of replication.
It has a multiple cloning site and a selectable marker gene, both
of which remain under the control of eukaryotic promoter and
polyadenylation sequences. These sequences are obtained from either
animal viruses (SV40, herpes simplex virus) or mammalian genes
(growth hormone, metallothionein). The promoter sequences
facilitate the transcription of cloned genes (at the multiple
cloning site) and the selectable marker genes. On the other hand,
the polyadenylation sequences terminate the transcription.
Collection and purification process of Recombinant proteins As the
recombinant proteins start accumulating in the host cells, it
becomes important to collect and purify them. This is a tricky
process since many times the recombinant protein is a foreign body
for the host cells and the enzyme machinery of the host cell
becomes activated to degrade the outside protein. One of the
strategies adopted is the use of bacterial strains deficient in
proteases or alternatively, the recombinant proteins are fused with
the native host proteins. The fusion proteins are resistant to
protease activity. Sometimes, the foreign proteins accumulate as
aggregates in the host organism which minimizes the
protease degradation. The best way out is to quickly export and
secrete out the recombinant proteins in to the surrounding medium.
The recovery and the purification of foreign proteins is easier a)
Gel and sponge technique- In this method, the gel (collagen) or
sponges (gelatin) are used which provides the matrix for the
morphogenesis and cell growth. The cells penetrate these gels and
sponges while growing. b) Hollow fibers technique- In this method,
hollow fibers are used which helps in more efficient nutrient and
gas exchange. In recent years, perfusion chambers with a bed of
plastic capillary fibers have been developed to be used for
histotypic type of cultures. The cells get attached to capillary
fibers and increase in cell density to form tissue like structures.
c) Spheroids The re-association of dissociated cultured cells leads
to the formation of cluster of cells called spheroids. It is
similar to the reassembling of embryonic cells into specialized
structures. The principle followed in spheroid cultures is that the
cells in heterotypic or homotypic aggregates have the ability to
sort themselves out and form groups which form tissue like
architecture. However, there is a limitation of diffusion of
nutrients and gases in these cultures.
d) Multicellular tumour spheroids- These are used as an in vitro
proliferating models for studies on tumour cells. The multicellular
tumour spheroids have a three dimensional structure which helps in
performing experimental studies related to drug therapy,
penetration of drugs besides using them for studying regulation of
cell proliferation, immune response, cell death, and invasion and
gene therapy. A size bigger than 500 mm leads to the development of
necrosis at the centre of the MCTS. The monolayer of cells or
aggregated tumour is treated with trypsin to obtain a single cell
suspension. The cell suspension is inoculated into the medium in
magnetic stirrer flasks or roller tubes. After 3-5 days, aggregates
of cells representing spheroids are formed. Spheroid growth is
quantified by measuring their diameters regularly. The spheroids
are used for many purposes. They are used as models for a vascular
tumour growth. They are used to study gene expression in a
threedimensional configuration of cells. They are also used to
study the effect of cytotoxic drugs, antibodies, radionucleotides,
and the spread of certain diseases like rheumatoid arthritis.
Organotypic cultures
These cultures are used to develop certain tissues or tissue
models for example skin equivalents have been created by culturing
dermis, epidermis and intervening layer of collagen simultaneously.
Similarly models have been developed for prostrate, breast etc.
Organotypic culture involves the combination of cells in a specific
ratio to create a component of an organ. BIOETHICS IN ANIMAL
GENETIC ENGINEERING There are some serious issues related to
genetic modification of animals using animal genetic engineering
techniques. One is not sure of the consequences of these genetic
modifications and the further interaction with the environment.
Proper clinical trials are also necessary before one can use it for
commercial purposes. In the recent past people have raised
objections on some of the methods used e.g. the transfer of a human
genes into food animals, use of organisms containing human genes as
animal feed. Some religious groups have expressed their concern
about the transfer of genes from animals whose flesh is forbidden
for use as food into the animals that they normally eat. Transfer
of animal genes into food plants that may be objectionable to the
vegetarians. Besides this, there are several other aspects of this
issue have to be sorted out.
a) What will be the consequences, if a modified animal will
breed with other domestic or wild animals thereby transferring the
introduced genes to these populations? b) What are the health risks
to human on consumption of genetically modified animals and
their products? c) With the production of disease resistant
animals, what will be the effect on ecology? d) There is also wide
spread concern about the risks of human recipients getting infected
with animal viral diseases after a xenotransplantation., which
might infect the population at large. e) There are also concerns
about the risk that drug resistance gene markers used in genetic
engineering procedures might inadvertently be transferred and
expressed. The need of the hour is to formulate clear guidelines
which should be followed while using genetic engineering techniques
in bio-medical research. e.g. products from transgenic organisms
should be clearly marked to give choice to people who follow
dietary restrictions due to religious beliefs. In fact all the
ethical and moral issues raised by some aspects of biotechnology
should be addressed by open discussion and dialogue.
CELL AND TISSUE ENGINEERING Tissue engineering refers to the
application of the principles of engineering to cell culture for
the construction of functional anatomical units- tissues/organs.
The aim of tissue engineering is nothing but to supply the various
body parts for the repair or replacement of damaged tissues or
organs. It is now possible to grow skin cells, blood cells cardiac
cells etc. by using the ability of stem cells to proliferate and
differentiate. During the last decade, the tissue culture work in
animals demonstrated that virtually any human tissue or organ can
be grown in culture. This became possible only after it became
known that the ability of cultured cells to undergo differentiation
can be restored. Skin was the first organ to be cultured in
artificial media and could be successfully used for transplantation
following serious skin burns. For past few years some of the
biotech companies like ATS (Advanced Tissue Science, USA),
Biosurface Technology (BTI, Cambridge) and Organogenesis, are
developing artificial skins to the stage of clinical trials. In the
field of tissue replacement, focus of attention is the Artificial
cartilage. As it is not vascularized, it is not rejected due to
immunogenic response. This will have lots of
implications in the treatment of sport related injuries and
diseases like arthritis. Design and engineering of tissues The
design and tissue engineering should essentially cause minimal
discomfort to the patient. The damaged tissues should be easily
fixed with the desired functions quickly restored. Another
important factor controlling the designing of tissue culture is the
source of donor cells. The cells from the patient himself, is
always preferred as it considerably reduces the immunological
complications. However under certain situations allogeneic cells
(cells taken from a person other than the patient) are also used.
The other important factors are the support material, its
degradation products, cell adhesion characteristics etc. It was
demonstrated in 1975 that human keratinocytes could be grown in the
laboratory in a form suitable for grafting. A continuous sheet of
epithelial cells can be grown now however there is still difficult
to grow TE skin with the dermal layer with all the blood
capillaries, nerves, sweat glands, and other accessory organs. Some
of the implantable skin substitutes which are tissue engineering
skin constructs with a limited shelf life of about 5 days are:
a) Integra TM A bioartificial material composed of
collagen-glycosaminoglycan and is mainly used to carry the seeded
cells. b) DermagraftTM- This is composed of poly glycolic acid
polymer mesh seeded with human
dermal fibroblasts from neonatal foreskins. c) ApligrafTM- It is
constructed by seeding human dermal fibroblasts into collagen gel
with the placement of a layer of human keratinocytes on the upper
surface. These tissue constructs integrate into the surrounding
normal tissue and form a good skin cover with minimum immunological
complications. The urothelial cells and smooth muscle cells from
bladder are now being cultured and attempts are on to construct TE
urothelium. Some progress has also been made in the repair of
injured peripheral nerves using tissue engineered peripheral nerve
implants. The regeneration of the injured nerve occurs from the
proximal stump to rejoin at distal stump. The regeneration process
requires substances like(a) Conduct material- The conduct material
is composed of collagenglycosaminoglycans, PLGA (poly lactic- co-
glycolicacid), hyaluronan and fibronectin and forms the outer
layer. (b) Filling material- The filling material contains
collagen, fibrin, fibronectin and agarose. This supports the neural
cells for regeneration. and
(c) Additives- A large number of other factors are also added
e.g. growth factors, neurotrophic factors such as fibroblast growth
factor (FGF), nerve growth factor (NGF). The other important
applications of tissue engineering are in gene therapy,
pseudo-organs and as model cell systems for developing new
therapeutic approaches to human diseases.The attempts are on to
create tissue models in the form of artificial organs using tissue
engineering. The artificial liver is being created using
hepatocytes cultured as spheroids and held suspended in artificial
support system such as porous gelatin sponges, agarose or collagen.
Some progress has been made in the area of creating the artificial
pancreas using spheroids of insulin secreting cells which have been
developed from mouse insulinoma beta cells. Three dimensional brain
cell cultures have been used for the study of neural myelination,
neuronal regeneration, and neurotoxicity of lead. The aggregated
brain cells are also being used to study Alzheimers disease and
Parkinsons disease. Thyroid cell spheroids are being used to study
cell adhesion, motility, and thyroid follicle biogenesis. (Table
8.2 page 155, gupta)TABLE DEPICTING THE TECHNOLOGICAL GOALS AND
AREAS OF RESEARCH IN TISSUE ENGINEERING
Growth of cells in three- dimensional systems
Delivery systems for protein therapeutics Cell cultivation
methods for culturing recalcitrant cells Expression of transgenic
proteins in transplantable cells To develop vehicles for delivering
transplantable cells Development of markers for tracking
transplanted cells Avoiding immunogenicity in transplantable cells
Development of in vivo and ex vivo biosensors for
monitoring cell behaviour during tissue production DOWNSTREAM
PROCESSING Downstream processing or downstreaming is the extraction
and purification of the desired end products of fermentation
processes. Such products might include cells, solvents or solutes.
Various processes are available for the separation of cells from
the fermentation broth in which they are grown, including
flocculation, filtration, centrifugation, sedimentation or
flotation. The procedure adopted depends on whether it is the
cells, or the solution surrounding them, that contains the desired
end products.