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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5992_Библиотеки_им_академика_М_И_Перельмана.pdf
Animal Cell and Tissue Biotechnology
65
Feeder Layers on Substrate
The substrate surface is a special type of monolayer of cells that are used partly, to supplement the
culture medium for growth and partly, for conditioning of the substrate by cell products. These
monolayers are also called as feeder layers, since they also feed the growing cultures. The feeder
layer may consist of mouse embryo fibroblasts, normal fetal intestine, etc. They are effectively
used for selective growth of breast and coelomic epithelium, central and peripheral neurons and
also for growing cells used for the production of transgenic animals through transfection.
Gas Phase for Tissue Culture
For growth of animal cell and tissue cultures, a gas phase consisting of oxygen and carbon dioxide
is also required, even though cultures may vary in their oxygen requirement. These gases also
influence pH and HCO
3
<
ion concentration of the culture.
Processing of Culture
The apparatus and liquids meant for tissue culture should be reserved separately for the purpose
and the glassware should be washed separately. The important steps involved in preparation
of culture and sterilization of glassware, equipment, reagents and media are described in the
following sections.
Glassware and Apparatus
1. The soiled glassware should not be allowed to dry. It has to be cleaned in water with sodium
hypochlorite to prevent microbial contamination. Automatic washing machines can also be
used for this purpose.
2. A nontoxic and effective detergent should be used.
3. After washing, glassware should be thoroughly rinsed with tap water followed by deionized
or distilled water and then dried upside down. The dried glassware is sterilized by dry heat (in
oven at 160°C for 1 h).
Reagents and Media
1. The reagents and components of media are procured in pure form.
2. They are sterilized either by moist heat, i.e. by autoclaving (if heat-stable) or by membrane
filtration (if heat-labile). During autoclaving (moist heating by steam), the container is kept
sealed (if borosilicate or polycarbonate) or the caps may be left slack (if soda glass) to avoid
breakage.
3. The liquid layer in container may reduce due to evaporation and should be restored with
sterile distilled water after autoclaving.
4. Alternatively, presterilized culture media available in market are also used.
Preparation of Animal Material
It is essential that contamination of any kind should be avoided during experimental manipulations,
by collecting tissue aseptically or sterilizing it after removal. The use of laminar flow cabinet or hood
Textbook of Pharmaceutical Biotechnology
66
facilitates the handling of material under aseptic conditions. The embryo or other contents of uterus
are free from contamination, whereas other adult tissues are contaminated with bacteria. The embryo
should be removed carefully under aseptic conditions, eliminating the possibility of any contamination.
The adult tissue is washed with a solution containing high concentration mixture of antibiotics (e.g. a
BSS containing 100 units of penicillin and 0.5 mg of streptomycin or neomycin per mL).
Disaggregation of Culture
A primary cell culture is obtained either by allowing cells to migrate from the tissue or by disaggregating
the tissue mechanically or enzymatically for the production of suspension of cells. The normal
untransformed cells can survive and proliferate easily to produce primary culture when attached to a
substrate. These cells are obtained by disaggregation achieved by one of the following techniques:
Physical disruption
It involves cutting the tissue into pieces.
Enzymatic digestion
Several enzymes are used for disaggregation, such as trypsin pronase, collagenase, elastase, pancreatin,
mucase, papain, etc. Enzymatic disaggregation is labour intensive and causes damage to cells.
Treatment with chelating agents
The chelating agents are used for production of cell suspensions from established cultures.
Epithelium tissues, which need Ca
++
and Mg
++
ions for their integrity, may be treated with chelating
agents, such as citrate and ethylenediaminetetraacetic acid (EDTA).
Isolation of Tissue
Freshney (1987) described various protocols for isolation of tissues, such as mouse embryo, hen’s
egg, human biopsy material, etc. The mechanical or enzymatic disaggregation gives a much higher
yield of representative cells in a short duration. For tissue isolation, the site is sterilized with 70%
ethanol and the tissue is removed aseptically. The isolated tissue is stored in a refrigerator before
transferring it to BSS or to a culture medium. The viable cells can be recovered from chilled tissue.
Enzymatic disaggregation
The embryonic tissue disperses more readily and gives higher yield than the newly born or adult. In
several tumours, the tissue being fragile is mostly destroyed during enzyme treatment, and viable
cells are obtained with great difficulty. The enzymatic disaggregation of the tissue under study is
possible by the following methods.
1. Trypsinization: Crude trypsin is the most common enzyme used for disaggregation.
Trypsinization is of two types: (1) warm trypsinization and (2) cold trypsinization. The latter
is effective for many tissues and is tolerated well by a number of cells. Its residual activity is
neutralized by serum of the medium or by a trypsin inhibitor (e.g. soybean trypsin inhibitor)
in case of a serum-free medium. The cells are exposed to warm enzyme (36.5°
C)
for a fixed
duration. The dissociated cells are collected every half an hour. The trypsin is removed by
Animal Cell and Tissue Biotechnology
67
centrifugation after complete disaggregation, which takes approximately 4 h. Alternatively,
cold trypsinization may be used that involves soaking of tissue in trypsin to allow penetration
of enzyme, followed by incubation at 36.5°C for a shorter period.
2. Disaggregation by collagenase: Sometimes, the disaggregation by trypsin is damaging for
epithelial cells or it is ineffective against fibrous tissues. The intracellular matrix contains
collagen. The enzyme collagenase is effective in the disaggregation of several normal and
malignant tissues, which may be sensitive to trypsin. Crude collagenase (with contamination
of nonspecific proteases) is often used with a firmly chopped tissue in complete medium.
When tissue is disaggregated, collagenase is separated by centrifugation, and the cells are
seeded and cultured at a high concentration.
Mechanical disaggregation
By this technique, the cell suspension is obtained more quickly as compared to enzymatic
disaggregation. The mechanical disaggregation is a force-based technique. The tissue is carefully
sliced, and the cells which spill out are collected. In this technique, the cells are either pressed
through the sieves of gradually reduced mesh or forced through a syringe and needle, or repeatedly
pipetted. This is useful in giving good yield of cells in a shorter time.
Separation of cells
The dissociated cells obtained as above are usually described as primary cells. They grow well
when seeded on culture plates at high density. These cells represent adherent primary cultures
in which the nonviable cells are removed at the first change of medium. The primary cultures
can also be maintained in suspension, wherein the nonviable cells are gradually diluted out on
commencement of cell proliferation. The nonviable cells can be separated from primary disaggregate
by centrifugation method.
Somatic Cell Fusion
The technique of somatic cell fusion is useful in the study of the control of gene expression
differentiation and in understanding the problem of malignancy, viral replication, gene mapping
and production of hybridomas for antibody production. Fusion of different types of cells has been
successfully achieved in animals to produce hybrid cells. The cell fusion in animals has been studied
within the body, e.g., myoblasts fusing to form multinucleate muscle fibres. They can also be fused
in vitro to
produce binucleate heterokaryons or uninucleate hybrid cells. The cell fusion may be
induced by polyethylene glycol (PEG). The removal of surface carbohydrates is also, sometimes,
required for successful cell fusion. Sendai virus is also often used to induce cell fusion.
The hybrid cells were obtained from mixed cultures of two different cell lines derived from
mouse. Later, cell fusion using different taxa was successfully attempted.
Tissue Culture
Primary techniques
This technique is used for cultivation of pieces of fresh tissue derived from the organism. In addition
to being used for embryo and organ culture, this technique is modified to become specialized
Textbook of Pharmaceutical Biotechnology
68
techniques. The different techniques are (1) slide or coverslip cultures, (2) carrel flask cultures and
(3) roller test tube cultures.
Slide or coverslip cultures
This is the oldest method of tissue culture being used, especially for morphological studies and
for cinemicrographic investigations. The slides or coverslip cultures are prepared by placing a
fragment of tissue onto a coverslip that is subsequently inverted over the cavity of a depression
slide. The advantages and limitations of this technique are outlined below.
Advantages
1. It is simple and relatively inexpensive technique.
2. The cells grow directly on coverslip and can be fixed and stained to make permanent slides.
3. The cells in living state are spread out in a manner suitable for microscopy and photography.
Limitations
1. The supply of oxygen and nutrients is rapidly exhausted, and the medium quickly becomes
acidic.
2. The sterility of cultures cannot be maintained for a long period.
3. Very small amount of tissues can be cultured at a time.
Different techniques of slide or coverslip cultures are as follows:
1. Single coverslip with plasma clot: This technique involves the following steps:
a. The medium is prepared in two parts: one containing 50% plasma in BSS and the other
with 50% embryo extract in serum.
b. Under aseptic conditions and with the use of a capillary pipette, one drop of plasma
containing solution is placed in the centre of each one, or more coverslips.
c. A fragment of tissue ( explant) is transferred to this drop without crushing the tissue.
d. The embryo extract containing solution is added to it and mixed thoroughly before clotting
starts, and then the explant is located.
e. The two small spots of petroleum jelly are placed using a glass rod near the concavity of a
depression slide, and the slide is inverted over the coverslip. A gentle pressure is applied,
so that jelly sticks to the coverslip.
f. The culture medium is allowed to clot.
g. The slide is turned over and the margins of coverslip are sealed with paraffin.
h. It is then labelled properly and incubated at 37°C.
The single coverslip cultures are useful for short-term studies. Therefore, they are difficult to handle
except by process of transfer.
2. Single coverslip with liquid medium: It covers the following steps:
a. The explants are prepared and placed in culture medium in a watch glass.
b. The explants are then drawn into the tip of a capillary pipette.
c. One explant is deposited in the centre of each coverslip.
Animal Cell and Tissue Biotechnology
69
d. The liquid medium is spread out in a very thin circular film with explant protruding above
the surface.
e. A depression slide with petroleum jelly is applied immediately and preparation turned over with
a quick flip to prevent the fluid from running into the crevice between the slide and coverslip.
f. The coverslip is sealed and the slide is incubated at 37°C, upright or inverted; the tissue
grows on the cover glass.
3. Double coverslip with plasma clot: This technique resembles the single coverslip method.
In this technique a large depression slide is used, and the entire preparation is attached to it by
petroleum jelly and wax in such a way that the small coverslip is not in contact with the slide
at any point. The technique includes the following steps:
a. A small drop of BSS is placed on a large coverslip.
b. A square or round coverslip is placed over BSS in the centre of large coverslip.
c. Thereafter, the steps enlisted above for single coverslip method are followed.
Due to limitations in use of single coverslip cultures, double coverslip cultures are
recommended.
It is desirable to leave the explant in its original location to obtain long-term tissue cultures.
This needs washing, feeding, patching and transfer of cultures. It involves the following
steps:
1. The seal is removed using razor blade.
2. The large coverslip is removed with small one still attached and flipped over it, so that
culture is uppermost in orientation.
3. The small coverslip is detached from the large coverslip using needle and forceps.
4. It is then transferred to a Columbia staining dish (Petri dish or watch glass may also be
used) containing BSS.
5. The small coverslip is removed and is placed on a large coverslip. It is required to be
carefully controlled.
6. The culture is fed by adding a drop of feeding solution (e.g. BSS:serum:50% embryo
extract = 1: 1: 1) to the small coverslip.
7. A clean depression slide is attached using petroleum jelly.
Carrel flask cultures
The Carrel flask technique is used for establishment of a strain from fresh explants of tissue, as it
has excellent optical properties for microscopic examination. The polystyrene culture flasks can
also be used for this purpose. Following two types of flask techniques are possible:
1. Thick clot culture: It allows rapid growth suitable for short-term cultures.
2. Thin clot culture: It can be maintained for a considerable period.
1. Preparation of fl ask cultures: It is achieved through the following steps:
a. Around six D-3.5 Carrel flasks are placed in a rack with their necks flamed and pointing to
the right.
Textbook of Pharmaceutical Biotechnology
70
b. A drop of plasma is placed on the floor of flask and spread out in a circle.
c. Desired number of explants are transferred to the plasma with the use of spatula and allowed
to clot.
d. After clotting of plasma and fixing of explants in position, extra medium is added.
e. Around 1.2 mL of dilute plasma is added for thick clots, whereas for thin clots 1.2 mL of
dilute serum is added instead of plasma and left for clotting.
f. The flasks are gassed with gas phase (5% CO
2
in air).
2. Renewal of medium: It is essential to remove the medium periodically, after which it is
replaced in flask cultures. The old fluid is drawn off with the help of a pipette. Around 1.2 mL
of fluid medium is added as replacement, and the flask is gassed with gas phase (5% CO
2
in
air).
3. Transfer of culture: For transfer, the culture grown in a flask culture is removed and cut into
pieces. These pieces are then used for replantation as usual.
The Carrel flask culture has the following advantages:
a. A large number of culture can be easily prepared.
b. A large amount of tissue can be grown with large quantity of medium.
c. The tissue can be maintained in the same flask for months or even for years.
Test tube culture
The operations of feeding, patching and transfer of culture are carried out as in other primary
explantation techniques. The cultures on plasma clots in test tube are prepared just like those in
flasks, but tissues can be grown on the wall of test tube without a plasma clot. The test tubes may
also be used for developing suspension cultures.
It is an economical technique, and it can be used for preparing a large number of cultures that
can be placed in stationary racks or roller drums. This technique, however, provides poor optical
property for microscopy. There is also high risk of contamination.
Organ Culture
It is possible to culture pieces of an organ or whole organ in vitro. In the technique of organ culture,
it is essential that the tissue is never disrupted or damaged. It requires careful handling. The media
used for growing organ cultures are generally the same as those used for tissue culture. The main
objective of this technique is to maintain the architecture of the tissue and direct it towards normal
development. The techniques of organ culture can be divided into two types: (1) those employing a
solid medium and (2) those employing liquid medium.
Culture of embryonic organs
Embryonic organ culture is easier to be established as compared to normal organs from adult animals.
Three techniques commonly used for establishment of embryo culture are described below.
1. Organ cultures on plasma clots: It involves the following steps:
a. The plasma clot is prepared by mixing 15 drops of plasma with 5 drops of embryo extract
in a watch glass.
Animal Cell and Tissue Biotechnology
71
b. The watch glass is placed on a pad of cotton wool in a Petri dish; cotton wool is kept moist
to prevent excessive evaporation from the dish.
c. A small, carefully dissected piece of tissue is placed on top of plasma clot in the watch glass.
The technique is now modified. A raft of lens paper or rayon net is used on which the tissue is
placed. The transfer of tissue can then be achieved by moving the raft easily. The excess fluid is
removed, and the net with the tissue is placed again on fresh pool of medium.
2. Organ cultures on agar: The medium solidified with agar is also used for establishment of
organ culture. The medium with agar provides the mechanical support for organ culture and
it does not liquefy. Embryonic organs generally grow well on this medium. This medium
consists of seven parts of 1% agar in BSS, three parts of chick embryo extract and three parts
of horse serum. Defined media with or without serum, but with agar, are also used.
The cultures of adult organs or parts from adult animal are more difficult to establish due to
their greater requirement of O
2
. A variety of adult organs (e.g. liver) have been cultured using
special media with a special apparatus (Towell’s type II culture chamber). Since the serum is found
to be toxic, serum-free media is also used. The special apparatus is devised, which permitted the
use of 95% O
2
.
Whole-Embryo Culture
In this technique, 40-hour-old embryos are used, and the development could be followed for another
24–48 h in vitro before the embryo dies.
The following steps are involved in the preparation of chick embryo culture:
1. A suitable defined medium is prepared and added to sterile watch glasses, placed on moist
absorbent cotton wool pads in Petri dishes (as for organ culture).
2. The hen’s eggs are incubated at 38°C for 42 h to provide about a dozen embryos.
3. The shell is wiped with alcohol and broken into a sterile evaporating dish, containing 50 mL
chick saline or BSS.
4. A circular cut is made into the vitelline membrane around the blastoderm using scissor, and
the latter is transferred to a Petri dish containing BSS .
5. The adherent vitelline membrane is removed with the aid of forceps, and the embryo is
examined under the microscope to determine the stage of development.
6. The blastoderm is spread on agar gel (ventral side down), and the excess BSS is removed.
7. The culture is incubated at 37.5°C.
Tissue Cultures in Biomedical Research
The cell culture methods are extensively used in both theoretical and applied genetic studies. The
various genetic applications are outlined below.
In karyological studies
The karyotype population of cells or an organism is the catalogue of the chromosomes of a typical
or an average cell. While karyotyping is a useful technique in experimental biology especially,
Textbook of Pharmaceutical Biotechnology
72
for identifying the origin of cell lines, its most important application in medicine is in the early
diagnosis of some congenital abnormalities. Mostly, these abnormalities result from the inheritance
of an unbalanced complement of chromosomes, such as Klinefelter’s syndrome (XXY), Turner’s
syndrome (XO) or Down’s syndrome (mongolism), wherein chromosome number 21 is represented
by 3 instead of 2. Some other anomalies such as translocations or deletions can also be recognized
using this technique of karyology.
In identification and hereditary metabolic disorders
The precise nature of many of the genetic diseases can be determined only by demonstrating the
metabolic abnormality, commonly the absence of an enzyme in tissue. Disorders such as absence of
catalase in acatalasia or of _-
D-galactose-l-phosphate, UDP glucose uridyltransferase in galactosaemia
or failure to decarboxylate branched chain keto acid in maple syrup urine disease can be demonstrated
in primary fibroblast cultures. The heterozygous locus, a deficiency of the transferase enzyme per
cell can also be measured. Some human genetic disorders that can be studied in this manner are
glycogen storage diseases, homocystinuria, Hurler’s syndrome, Hunter’s syndrome, Niemann-Pick
disease, I-cell disease, orotic aciduria, Refsum’s disease, xeroderma pigmentosa, etc.
In somatic-cell genetics
The studies involving somatic cell are made primarily with the following objectives:
1. For understanding behaviour of heterokaryons, following fusion of parent cells with different
properties
2. For knowing the behaviour of hybrid cells derived from heterokaryons with the objective of
eliciting information about interaction between different genes
3. For mapping loci on different chromosomes
In biomedicine
The study of the relationships between viruses and cells has been greatly supplemented with the
use of tissue culture methods. These techniques are also useful for detection and identification of
viruses from the suspected cases. In the recent years, the significance has been in the development
of vaccine against poliomyelitis that is made from the viruses grown in tissue culture. In addition to
viruses, other intracellular parasites that are grown in tissue culture and studied include rickettsiae,
mycobacteria (especially tubercle and leprosy bacilli), lepraemurium, parasitic protozoans, etc.
The ability of tissue cultures to support the growth of viruses and to reveal their presence by
lesions (that are in some cases specific) has been applied in virology for the following purposes:
1. Study of host–parasite relationship
2. Detection and identification of viruses
3. Production of viruses for vaccine manufacture
The tissue culture techniques have been very useful in understanding of cancer. The culture of
canine lymph sarcoma and the development of quantitative assay procedures for viruses responsible
for tumours—stimulated by the exploitation of cell culture methods by virologists—are significant
achievements in biomedical sciences.
Animal Cell and Tissue Biotechnology
73
QUESTIONS
1. Write a short note on maintenance of animal cell culture.
2. Give the difference between batch and continuous culture.
3. Explain the steps involved in the establishment of animal cell culture.
4. Explain the techniques used for in vitro animal cell culture.
5. Write the different phases of bacterial growth curve.
6. Discuss methods of single-cell culture techniques and their applications.
7. Write about sterilization methods used in tissue culture works.
8. Write a note on natural culture media for animal cell culture media.
9. Write the advantage and disadvantage of animal cell culture media.
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