Cell culture involves growing cells outside of their natural environment under controlled conditions. The document discusses the history and development of cell culture techniques. It explains the process of explant culture where a small piece of tissue is used to initiate a primary culture. The requirements for maintaining cell cultures such as appropriate nutrients, pH, temperature and regular sub-culturing to prevent overgrowth are also summarized. The major cell culture applications in areas like disease research, toxicology testing and genetic engineering are highlighted in brief.

1. CELL CULTURE AND CELL LINES AND ITS IMPLICATION
IN FISHERY
A SEMINAR ON
BY:
ASIK IKBAL
3RD YR B.F.Sc , WBUAFS

2.  Cell culture is the complex process by which cells are
grown under controlled conditions, generally outside of
their natural environment.
 Cell culture refers to a culture derived from dispersed
cells taken from original tissue, from a primary culture,
or from a cell line or cell strain by enzymatic,
mechanical, or chemical disaggregation.
 They require------- ongoing care,
------- adequate nutrition,
------- a proper environment and
------- regular checkups.
Introduction
Cell culture

3.  Fish cells removed from tissues, will continue to grow if
supplied with the appropriate nutrients and conditions.
 When it is carried out in a laboratory, the process is
called Cell Culture.
 cell culture became a common laboratory technique in
the mid-1900s, but the concept of maintaining live cell
lines separated from their original tissue source was
discovered in the 19th century.
 However, there are also cultures of plants, fungi and
microbes, including viruses, bacteria and protists.
Introduction

4. History
• The 19th-century English physiologist Sydney Ringer developed salt
solutions containing the chlorides of sodium, potassium, calcium and
magnesium suitable for maintaining the beating of an isolated animal
heart outside of the body.
• In 1885, Wilhelm Roux removed a portion of the medullary plate of
an embryonic chicken and maintained it in a warm saline solution for
several days, establishing the principle of tissue culture.
• Cell culture techniques were advanced significantly in the 1940s and
1950s to support research in virology.
• The research of fish cell culture has developed rapidly since Wolf and
Quimby established the first fish cell line RTG-2 in 1960s for the
first time.
• After then, fish cell culture has become an essential research
technology which has been used extensively.

5. MAJOR DEVELOPMENT’S IN CELL CULTURE TECHNOLOGY
First development was the use of antibiotics which
inhibits the growth of contaminants.
Second was the use of trypsin to remove adherent
cells to subculture further from the culture vessel.
Third was the use of chemically defined culture
medium.

6. Areas where cell culture technology
is currently playing a major role.
 Model systems for
Studying basic cell biology, interactions between disease
causing agents and cells, effects of drugs on cells, process and
triggering of aging & nutritional studies.
 Toxicity testing
 Cancer research
 Virology
 Genetic Engineering
 Gene therapy
Why is cell culture used for?

7. Tissue culture
 In vitro cultivation of organs, tissues & cells at defined
temperature using an incubator & supplemented with a
medium containing cell nutrients & growth factors is
collectively known as tissue culture.
 Different types of tissues grown in culture include
connective tissue elements such as fibroblasts, skeletal
tissue, cardiac tissue, epithelial tissue (liver, skin,
kidney) and many different types of tumor cells.

8. Primary culture
 Cells, when surgically or
enzymatically removed from an
organism and placed in suitable culture
environment, will attach and grow and
it is called as primary culture.
 Primary cells have a finite life span.
 Primary culture contains a very
heterogeneous population of cells.
 Sub culturing of primary cells leads to
the generation of cell lines.
 Lineage of cells originating from the
primary culture is called a cell strain.
Cell culture

9. Continuous cell lines
 Most cell lines grow for a limited number of generations
after which they ceases.
 Cell lines which either occur spontaneously or induced
virally or chemically transformed into continuous cell
lines.
 Characteristics of continuous cell lines
-smaller, more rounded, less adherent with a higher
nucleus /cytoplasm ratio
-Fast growth and have aneuploid chromosome number.
-reduced serum and anchorage dependence and grow
more in suspension conditions.
-ability to grow upto higher cell density.
-different in phenotypes from donor tissue.
-stop expressing tissue specific genes.
Established human cell lines
Fish cell lines

10. On the basis of morphology (shape &
appearance) or on their functional
characteristics, they are divided into three.
 Epithelial like- attached to a substrate and
appears flattened and polygonal in shape.
 Lymphoblast like- cells do not attach remain
in suspension with a spherical shape.
 Fibroblast like- cells attached to an substrate
appears elongated and bipolar.

11. Types of cell Cultures
 Cell cultures may contain the following three types of cells:
 1. Stem cells,
 2. Precursor cells and
 3. Differentiated cells
 Stem cells- they are undifferentiated cells which can differentiate under correct
inducing conditions into one of several kinds of cells; different kinds of stem
cells differ markedly in terms of the kinds of cells they will differentiate into.
 Precursor cells- these cells are derived from stem cells, are committed to
differentiation, but are not yet differentiated; these cells retain the capacity for
proliferation.
 Differentiated cells- they usually do not have the capacity to divide.
 Some cell cultures, e.g., epidermal keratinocyte cultures, contain all the three
types of cells.

12.  Cell cultures can be grown as:
 Cells need a surface or substrate to
adhere for proliferation.
 Cells that are unable to adhere to a
substrate are unable to divide, i.e., their
growth is anchorage dependent.
 The various kinds of substrates used in
cell cultures are(1) glass, (2) plastics, (3)
metals. Cell culture

13. Suspension culture
• At this stage, cells are usually heterogeneous but still closely
represent the parent cell types as well as in the expression of tissue
specific properties.
• After several sub-cultures onto fresh media, the cell line will either
die out or 'transform' to become a continuous cell line.
• Such cell lines show many alterations from the primary cultures
including change in morphology, chromosomal variation.
• Fish cells can be grown either in an unattached suspension culture or
attached to a solid surface.
• It has been successfully developed to quite large bioreactor volumes,
with successful production of viruses and therapeutic proteins.

14. Types of Suspension culture
 Suspension cultures are of following types:
 1. Batch Culture (fixed medium volume; as the cell grow,
medium is gradually depleted; eventually cells cease to divide),
 2. Fed Batch Culture (gradual addition of fresh medium leading
to an increase in culture volume),
 3. Semi-continuous Batch Culture (at regular intervals, a
constant fraction of the culture, including cells, is withdrawn
and an equal volume of fresh medium is added to the culture),
 4. Perfmion Culture (at regular intervals, a constant volume of
spent medium, without cells, is withdrawn and an equal volume
of fresh medium is added); and
 5. Continuous-flow Culture (continuous withdrawl of culture
along with cells and addition of equal volume of fresh medium
so that the culture is maintained in a steady state).

15. Media and supplements used in fish cell
culture
 The nutrient media used for culture of animal
cells and tissues must be able to support their
survival as well as growth, i.e., must provide
nutritional, hormonal and stromal factors.
The various types of media used
for tissue culture may be
grouped into two broad categories:
1. Natural Media and
2. Artificial Media.
Culture media

16. Natural media
 These media consist solely of naturally occurring
biological fluids and are of the following three types:
 (1) cagula or clots,
 (2) biological fluids and
 (3) tissue extracts.
 Biological Fluids----various biological fluids used as
culture medium (e.g., amniotic fluid, ascitic and
pleural fluid, aqueous humour from eye, insect
haemolymph, serum etc.). Serum is the most widely
used biological fluids.

17. Artificial Media
 Different artificial media have been devised to serve
one of the following purposes:
 (1) immediate survival (a balanced salt solution, with
specified pH and osmotic pressure is adequate),
 (2) prolonged survival (a balanced salt solution
supplemented with serum, or with suitable
formulation of organic compounds),
 (3) indefinite growth, and
 (4) specialized functions.

18. Artificial Media
 The various artificial media developed for
cell cultures may be grouped as:
 (i) serum containing media
 (ii) serum-free media,

19. Serum Containing
Media
 The various defined media, (e.g., Eagle's minimum
essential medium etc.) when supplemented with 5-
20% serum are good nutrient media for culture of most
types of cells.
 It provides the basic nutrients for cells; the nutrients
are present both in the solution as well as are bound to
the proteins.
 A major role of serum is to supply proteins
(e.g,fibrobnectin), which promote attachment of cells to
the substrate. It also provides spreading factors that
help the cells to spread out before they can begin to
divide.

20. Serum-free
media
Various defined serum free media is used due to
 1. Improved reproducibility of results
 2.variation due to batch change of serum is
avoided.
 3. Easier downstream processing of products
from cultured cells.
 4. Toxic effects of serum are avoided.
 5. Biassays are free from interference due to
serum proteins.

21. Serum-free
media
 Specific media like Leibovitz L15 is used
to eliminate the need of adding CO2 and
NaHCO3.
 After the initiation of primary culture,
fish serum is added at 1% final
concentration.

22. Basic Constituents of media
Inorganic salts
Carbohydrates
Amino Acids
Vitamins
Fatty acids and lipids
Proteins and peptides

23. Basic equipments used in cell culture
Laminar cabinet
Incubation facilities
Refrigerators
Microscope
Tissue culture ware

24. Buffering Systems
•Most cells require pH conditions in the range 7.2 - 7.4
and close control of pH is essential for optimum culture
conditions (however there are major variations to this
optimum).
•Fibroblasts prefer a higher pH (7.4 - 7.7) whereas,
continuous transformed cell lines require more acid
conditions pH (7.0 - 7.4).

25. Buffering Systems
•Regulation of pH is particularly important immediately
following cell seeding when a new culture is
establishing and is usually achieved by one of two
buffering systems;
•A "natural" buffering system where gaseous CO2
balances with the CO3 / HCO3 content of the culture
medium and
•Chemical buffering using a zwitterions.

26. Explant culture
• It is a technique used for the isolation of cells
from a piece or pieces of tissue. Tissue harvested
in this manner is called an explant.
• It can be a portion of the shoot, leaves, or some
cells from a plant, and can be any part of the
tissue from an animal.
• Primary cultures are derived directly from normal
animal tissue and cultured either as an explant
culture or dissociation into a single cell
suspension by enzyme digestion.

27. Explant Preparation
 Preparation of donor fish:
 The donor fish is usually starved for a day or two
to reduce the possibility of gross contamination
from feces and unconsumed feed.
 During this period, fish is allowed to swim in
well aerated autoclaved water for reducing the
microbial load adhered on to the skin and gills,
then sacrificed by plunging in ice for 10-15 min.
 Adequate care should be taken to minimize the
possible routes of contamination.

28. Explant Preparation
 Decontamination:
 The decontaminating solutions includes chlorine
solution (500 ppm), 70% ethanol, iodophore solution
(0.5 w/v iodine) etc. are used.
 Strong disinfectants are avoided because they damage
the tissue.
 The commonly used antibiotics are penicillin (400
IU/ml) and streptomycin (400 μg / ml) with an anti-
fungal amphotericin B (10 μg/ml).
 The tissue of interest is aseptically picked up and
washed three to five times with the antibiotic solution.

29. Explant Preparation
 Dissection and / or Disagregation: Two major methods for
initiating a primary culture followed are given below.
 (i) Tissues are disaggregated into its component cells. This is
achieved by enzymatic digestion using trypsin or collagenase
supplemented with EDTA.
 These enzymes digest the extra celluar matrix and EDTA
chelates divalent cations like Ca2+ and Mg2+ required for the
integrity of the matrix.
 The tissue was also dispersed by mechanical means like
slicing, sieving, forcing through a needle and repeated
pipetting.
 The collected cells are then seeded in the vessel at 5 x 105
cells per ml of medium.

30. Explant Preparation
 (ii) The required tissue is picked up and cut in small pieces to
prepare explants of 1 mm3 size and planted in culture dishes.
 1 or 2 fragments of tissue are seeded in 1 cm2 area.
 The caudal fin, heart and gills are aseptically excised from
fingerlings are rinsed individually with phosphate-buffered
saline (PBS), 70% ethanol and Iodine antiseptic (0.5% w/v
iodine).
 The head and gut of the fry should be carefully discarded
while the remaining tissue mass is washed as above.
 Explants of 1mm3 sizes are prepared and washed thrice with
PBS containing antibiotics for 5-10min.

31. Culture of cells following
seeding
 The explants are seeded in 25 cm2 tissue culture
flask and kept semi dry for a few minutes.
 The adherence of explants is accomplished by
incubation with 0.5 mL of PBS at 28° C. After 8-10
hrs, L-15 (Leibovitz) is added gently. 50% of the
media is recommended to be exchanged once in
every 3 days. Daily observations are made using an
inverted microscope. The dispersed cells adhere on
the culture substrate and start to proliferate.

32. Culture of cells following
seeding
 Suspended dead cells can be removed by
subsequent medium exchange. The optimum pH
should be maintained nearly 7.4 and incubation
temperature should be maintained 22-28° C for
culture of fish cells.
 Suspension cells are anchorage-independent e.g.
blood cells.
 Transformed cell lines either grows as monolayer
or as suspension.

33. Sub-culturing
• Once the available substrate surface is covered by cells (a
confluent culture) growth slows & ceases.
• Cells to be kept in healthy & in growing state sub-cultured or
passaged has been done.
• Enzyme such as trypsin, dipase, collagenase in combination
with EDTA breaks the cellular glue that attached the cells to
the surface.
• Cells intolerant to trypsin can be scraped using a cell scraper
or dispersed by rocking followed by gentle pipetting.
• Detached cells can then be distributed to 2 to 4 flasks
containing fresh medium depending on the split ratio required.

34. Growth pattern
 Cells initially goes through a
quiescent or lag phase that depends
on the cell type, the seeding density,
the media components, and previous
handling.
 The cells will then go into
exponential growth where they have
the highest metabolic activity.
 The cells will then enter into
stationary phase where the number of
cells is constant, this is characteristic
of a confluent population.
Cell Culture of fin of L. rohita

35. Maintenance
Cultures should be
examined daily,
observing the
morphology, the
color of the
medium and the
density of the cells.
Epithelial cell culture

36. Harvesting
 Cells are harvested when they
reach a population density which
suppresses growth.
 Ideally, cells are harvested when
they are in a semi-confluent state
and are still in log phase.
Cultured cells

37.  Cell viability is determined by staining the cells with trypan
blue.
 As trypan blue dye is permeable to non-viable cells or death
cells whereas it is impermeable to viable cells.
 The cells are stained with trypan dye and loaded to
haemocytometer and calculation of % of viable cells is done.
Number of unstained cells x 100
% of viable cells=
total number of cells
Cell viability

38. Cell culture contaminants of two types-
Chemical-difficult to detect caused by
endotoxins, plasticizers, metal ions or traces of
disinfectants that are invisible.
Biological-causes visible effects on the culture
they are mycoplasma, yeast, bacteria or fungus
or also from cross-contamination of cells from
other cell lines.

39.  Cytotoxicity causes inhibition of cell growth.
 It effects on the morphological alteration in the
cell layer or cell shape.
 Cytotoxicity is determined by substituting
materials such as medium, serum, supplements
flasks etc. at a time.

40. BASIC ASEPTIC CONDITIONS TO BE MAINTAINED
IN THE CELL CULTURE LABS
 If working on the bench uses a Bunsen flame to
heat the air surrounding the Bunsen.
 Swab all bottle tops & necks with 70% ethanol.
 Flame all bottle necks & pipette by passing very
quickly through the hottest part of the flame.
 Avoiding placing caps & pipettes down on the
bench; practice holding bottle tops with the little
finger.
 Work either left to right or vice versa, so that all
material goes to one side, once finished, Clean up
spills immediately & always leave the work place
neat & tidy.

41. Applications for Fish cell
cultures
 To investigate the normal physiology or biochemistry
of cells. For instance, studies of cell metabolism.
 To test the effect of various chemical compounds or
drugs on specific cell types (normal or cancerous
cells, for example).
 To study the sequential or parallel combination of
various cell types to generate artificial tissues.
 Therapeutic proteins can be synthesized in large
quantities by growing genetically engineered cells in
large-scale cultures.
 Creation of viral vaccines from large scale cell
cultures.
 Cytotoxicity and genotoxicity studies.

42. Advantages of cell culture
 The major advantage of using cell culture is the consistency
and reproducibility of results that can be obtained from
using a batch of clonal cells.
 The materials are cheap and easy to obtain.
 The experimental condition could be controlled accurately.
 Can control all external factors.
 Can easily test what the cells are doing.
 Cells are easy to manipulate and propagate.
 All of the cells are the same hence results of experiments
will be consistent.
 Cheaper to maintain.

43. Safety aspect in cell culture
•Possibly cultures should be kept free of antibiotics.
•The same media bottle for different cell lines never
be used. If caps are dropped they are replaced with
new ones.
•Necks of glass bottles prefer heat at least for 60 secs
at a temperature of 20°C.
•Switch on the laminar flow cabinet 20 minutes prior
to start working.
•Cell cultures which are frequently used should be
subcultured & stored as duplicate strains.

44.  After a period of continuous growth, cell characteristics can
change. Cells can also adapt to different culture environments by
varying the activities of their enzymes.
 Expertise handling is needed to check chemical, microbial and
cross contamination.
 Require a control environment in the workplace for incubation,
pH control containment and disposal of biohazards.

45.  Quantity and cost involvement is more.
 Genetic instability like heterogeneity and variability may appear.
 Sometimes the phenotypic characteristics of the tissue may get
lost due to dedifferentiation and adaptation.
 If the differentiated properties are lost, it is difficult to relate the
cultured cell with the functional cell in the tissue.

46.  As the anatomy and physiology of fish is complex, there are
many different cells and proteins which are interacting
continuously for normal body functioning.
 Being complex in nature, these events are difficult to watch
individually in vivo.
 Moreover, fish being delicate are usually harmed and get
stressed while observing biological events.
 Hence it is necessary to develop a parallel cell observation
system particularly in vitro in nature to which a cell culture
system strongly supplements.
Conclusion

47. References
 Basics of Cell Culture--- Paras Yadav1, Annu Yadav1, P.
Kumar1, J.S. Arora1, T.K.Datta1, S. De1, S.L. Goswami1,
Mukesh Yadav2, Shalini Jain3, Ravinder Nagpal4 and
Hariom Yadav3
An Overview Of Cell Culture In Fish---Mohan R.
Badhe, Priyanka C. Nandanpawar (CIFE, Mumbai).
Cell culture --- From Wikipedia.
www.google.com