Tissue culture involves growing cells and tissues outside of their natural environment in laboratory conditions. Some key points: - Tissue culture originated in the late 19th/early 20th century with experiments maintaining animal and plant cells. - It allows cloning of cells with the same genotype and study of cell/tissue growth and behavior. - Primary cultures have a finite lifespan while continuous cell lines are immortalized and can proliferate indefinitely. - Cells must be subculture when confluent to maintain healthy growth, and can be cryopreserved for long-term storage. - Proper aseptic technique and controlled conditions like temperature, pH, gas exchange are required to prevent contamination.

1. CELL AND TISSUE CULTURE

2. WHAT IS IT?
 Tissue culture is the term used for “the
process of growing prokaryotic, eukaryotic or
plant cells artificially in the laboratory”
 But in practice it refers to the culturing of
cells derived from animal cells.
 Tissue culture involves both plant and
animal cells
 Tissue culture produces clones, in which all
product cells have the same genotype
(unless affected by mutation during culture).

3. HISTORY
 1880 - Arnold showed that leucocytes can divide outside the
body
 1885: Roux maintained embryonic chick cells in saline
 1903 - Jolly studied the behavior of animal tissue explants
immersed in serum, lymph, or ascites fluid.
 1907 - Ross Granville Harrison cultured frog tadpole spinal chord
in a lymph drop hanging from a cover slip of a cavity slide.
 1913 - Carrel developed a complicated methodology for
maintaining cultures free of contamination
 1965: Harris & Watkins successfully fused human and mouse cells
by virus
 1975: Kohler & Milstein produced the first Hybridomas capable
of secreting monoclonal antibodies. 8/5/2022
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4. HISTORY
 Wilhelm Roux in 1885 for the first time
maintained embryonic chick cells in a
warm saline solution in cell culture for 7
days.
 Cell culture was first successfully
undertaken by Ross Harrison in 1907.
He cultured frog neuroblasts in a lymph
medium. He works on nerve-cell
outgrowth.

5.  Tissue culture had its origins at the
beginning of the 20th century with
the work of Gottleib Haberlandt
(plants) and Alexis Carrel
(animals)
 Alexis Carrel create the "perfusion
pump," which allowed living
organs to exist outside of the body
during surgery.
Carrel
Haberlandt

6. 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.

7.  A more recent advance is the use of plant and
animal tissue culture along with genetic
modification using viral and bacterial vectors and
gene guns to create genetically engineered
organisms

8. TISSUE CULTURE
 Tissue culture: includes cell culture and
organ culture
 Cell culture: dispersed cells taken from
original tissue, from a primary culture, or
from a cell line by enzymatic, mechanical, or
chemical disaggregation.
 organ culture : a three-dimensional culture of
undisaggregated tissue retaining some or all
of the histological features of the tissue in
vivo

9.  Histotypic culture :implies that cells have
been reaggregated or grown to re-create a
three-dimensional structure with tissue like
cell density , e.g. overgrowth of a monolayer
in a flask or dish, reaggregation in suspension
over agar
 Organotypic culture: implies the same
procedures but recombining cells of different
lineages, e.g., epidermal keratinocytes in
combined culture with dermal fibroblasts, in
an attempt to generate a tissue equivalent.

10. WHAT DO YOU NEED TO DO
IT?
 Source of cell material
-freshly prepared
-stock of cell line
-bacterial culture

11. SUITABLE CONTAINER
 Simple flask
 Sophisticated fermenter with
computer-controlled
monitoring

12. GROWTH MEDIUM
 Glucose
 Water
 Amino acids:
 Salts

13. ANIMAL SERUM
 Foetal Bovine Serum
 Essential for animal cell proliferation
 5% - 10% of growth media

14. OPPORTUNITY FOR GAS
EXCHANGE
 Oxygen
 Carbon dioxide

15. INDICATOR
 Waste products causes change in pH
 Use indicator like phenol red
 Changes from orange to yellow

16. CONTROL OF TEMPERATURE
AND PH
 37.0 OC
 pH 7.2-7.4

17. METHOD FOR MEASURING CELL
NUMBER
 Counting cell numbers in culture
(haemocytometer)
 Measure optical density in spectrophotometer

18. STERILISATION
 Antibiotics
 Sterilisation
a. Hot air Oven
b. Autoclave
c. Filtration

19. CELLS ARE EITHER….
 Anchorage – dependant
 Anchorage - independant

20. ANCHORAGE – INDEPENDANT
CELLS
 Cells associated with body fluid
-blood cells
 Grown in suspension
 Will eventually need subculturing

21. ANCHORAGE – DEPENDANT
CELLS
 Most animal derived cells
 Adhere to bottom of a flask and form a monolayer
 Eventually cover entire surface of substratum
(confluence)
 Proliferation then stops
 Need to subculture cells at this point (remove to fresh
medium)
 Proliferation can begin again

22. 2 MAIN CATEGORIES OF ANIMAL
CELL CULTURES….
 Primary culture
 Continuous cell line

23. PRIMARY CULTURES
 Taken from fresh tissue
 Limited life span in culture
 Treated by proteolytic enzyme (Trypsin)
 Separate into single cells
-epithelial cells
-fibroblasts

24. PRIMARY CULTURE
 Cells when surgically or enzymatically removed from an
organism and placed in suitable culture environment
will attach and grow are 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
 Finite Cell lines have limited life span, they passage
several times before they become senescent
 Cells such as macrophages and neurons do not divide in
vitro so can be used as primary cultures
 Lineage of cells originating from the primary culture is
called a cell strain

25. CONTINUOUS CELL LINES
 Cell lines which either occur spontaneously or induced
virally or chemically transformed into Continous cell lines
 Produce immortalised cell lines
 Characteristics of continous cell lines
 smaller, more rounded, with a higher nucleus /cytoplasm
ratio
 Often lose their anchorage-dependence
 lose sensitivity to factors associated with growth control
 Fast growth and have ability to grow upto higher cell
density
 Do not have contact inhibition

26. CONTINUOUS CELL LINE
 Cell lines are neoplastic
 associated with an altered xsome pattern i.e.
Aneuploid chromosome number
 reduced serum and anchorage dependence and grow
more in suspension conditions
 different in phenotypes from donar tissue
 stop expressing tissue specific genes

27. Cell
line
Species
of origin
Tissue of
origin
Cell
morphology
Growth in
suspension?
3T3 Mouse Connective Fibroblast No
CHO Chinese
Hamster
Ovary Epithelial Yes
BHK21 Syrian
Hamster
Kidney Fibroblast Yes
HeLa Human Cervical
Carcinoma
Epithelial Yes

28. WHY 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
have to be sub-cultured or passaged
 It’s the passage of cells when they reach to
80-90% confluency in flask/dishes/plates
 Enzyme such as trypsin, dipase, collagenase in
combination with EDTA breaks the cellular
glue that attached the cells to the surface

29. ADHERENT CELLS
 Cells which are anchorage dependent
 Cells are washed with PBS (free of ca & mg ) solution.
 Add enough trypsin/EDTA to cover the monolayer
 Incubate the plate at 37 C for 1-2 mts
 Tap the vessel from the sides to dislodge the cells
 Add complete medium to dissociate and dislodge the cells
 with the help of pipette which are remained to be adherent
 Add complete medium depends on the subculture
 requirement either to 75 cm or 175 cm flask

30. SUSPENSION CELLS
 Easier to passage as no need to detach them
 As the suspension cells reach to confluency
asceptically remove 1/3rd of medium
 Replaced with the same amount of pre-
warmed medium

31. FREEZING CELLS FOR
STORAGE
 Remove the growth medium, wash the cells by PBS and remove
the PBS by aspiration
 Dislodge the cells by trypsin-versene
 Dilute the cells with growth medium
 Transfer the cell suspension to a 15 ml conical tube, centrifuge
at 200g for 5 mts at RT and remove the growth medium by
aspiration
 Resuspend the cells in 1-2ml of freezing medium
 Transfer the cells to cryovials, incubate the cryovials at -80 C
overnight
 Next day transfer the cryovials to Liquid nitrogen

32. WORKING WITH CRYOPRESERVED
CELLS
 Vial from liquid nitrogen is placed into 37 C water bath, agitate vial
continuously until medium is thawed
 Centrifuge the vial for 10 mts at 1000 rpm at RT, wipe top of vial
with 70% ethanol and discard the supernatant
 Resuspend the cell pellet in 1 ml of complete medium with 20%
FBS and transfer to properly labeled culture plate containing the
appropriate amount of medium
 Check the cultures after 24 hrs to ensure that they are attached to
the plate
 Change medium as the colour changes, use 20% FBS until the
cells are established

33. CELL VIABILITY
 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 this dye
 Stain the cells with trypan dye and load to
haemocytometer and calculate % of viable cells
% of viable cells= Nu. of unstained cells x 100
total nu. of cells

34. BASIC ASEPTIC CONDITIONS
 If working on the bench use 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

35. SAFETY ASPECT IN CELL CULTURE
 Possibly keep cultures free of antibiotics in order to be
able to recognize the contamination
 Never use the same media bottle for different cell lines.
If caps are dropped or bottles touched unconditionally
touched, replace them with new ones
 Necks of glass bottles prefer heat at least for 60 sec at a
temperature of 200 C
 Switch on the laminar flow cabinet 20 min prior to start
working
 Cell cultures which are frequently used should be sub
cultered & stored as duplicate strains

36. OTHER KEY FACTS…….?
 Use actively growing cells that are in their log phase
of growth, which are 80-90% viable
 Keep exposure to trypsin at a minimum
 Handle the cells gently. Do not centrifuge cells at high
speed or roughly re-suspend the cells
 Feeding & sub culturing the cells at more frequent
intervals then used with serum containing conditions
may be necessary
 A lower concentration of 104cells/ml to initiate
subculture of rapidly growing cells & a higher
concentration of 105cells/ml for slowing growing cells

37. ADVANTAGES OF TISSUE
CULTURE
 Animal experimentation can be avoided
 Behavior of cells is easily observed and regulated.
 Cells are homogenous.
 Optimizes growth pattern
 Enables control of the extracellular environment
 Allows monitoring of various elements and
secretions without interference from other
biological molecules
 Cost effective, as less quantities of reagents are
required as compare to in vivo system

38. DISADVANTAGES OF TISSUE
CULTURE
 Cells are devoid of in vivo interactive environment
 Needs controlled physiological and physiochemical
condition
 Productions of unwanted proteins due to de-
differentiation of cells in artificial condition
 Unstable aneuploid chromosomes
 Interpretation of the behavior of the cell needs expert
 Loss of phenotypic characterstic
 Expertise is needed

39. INVESTIGATION OF THE NORMAL
PHYSIOLOGY AND
BIOCHEMISTRY OF CELLS
 The primary impetus for the development of cell
culture was to study, under the microscope,
normal physiological events of cells.
 Haberlandt (1902) stated that the in vitro-culture
techniques for plants were developed primarily to
facilitate basic physiological research.

40.  Harrison (1907) developed his culture to study
the development of nerve fibers.
 Animal or plant cell, when removed from tissues
and supplied with the appropriate nutrients and
conditions, grows and acts as independent unit,
much like a microorganisms such as a bacterium or
fungus.

41. WHY IS CELL CULTURE USED FOR
Areas where cell culture technology is currently playing
a major role.
 Model systems for Studying basic cell biology,
 Toxicity testing
 Cancer research
 Genetic Engineering
 Gene therapy
 Karyotyping studies
 Replacement of damaged tissue and cells
 Virology
 Production of medicinal & commercial proteins

42. USE OF TISSUE CULTURES IN
TOXICITY TESTING
 Mammalian cell cultures can be a suitable
alternative for the use of whole animal tests
to establish the potential toxicity of
compounds.
 This due to many reasons:
 1- They can overcome the disadvantages of
the whole animal tests including:
 High costs.
 Variability of results.

43. CONT…….
 2- Growing moral objections to the use of
animals in toxicity testing.
 3- Cell culture tests are rapid, allow more
efficient screening of novel compounds and
sometimes can allow the identification of
metabolic targets of inhibition.

44. CANCER RESEARCH
 Tumors can be produced artificially
 Anti cancerous compounds can be
tested in in vitro developed tumors
and on cancerous cell lines

45. USE OF TISSUE CULTURES FOR
PRODUCTION OF
BIOLOGICAL PRODUCTS
 A) Production of vaccines:
 Two factors stimulated the use of tissue cultures for
vaccine production:
 The ability to grow viruses in cell cultures.
 Current egg-vaccine production requires long time
(9 months) that hinder the response to unanticipated
demands.

46. PRODUCTION OF VACCINES
CONT…..
 In (1949), Enders discovered that the
poliomyelitis virus could be grown from
primary monkey cells in culture.
 The polio vaccine, produced in 1954, was the
first human vaccine to be produced using
large-scale cell culture techniques.
 Animal cell technology is considerably
developed for the production of a range of
human and veterinary viral vaccines against a
variety of diseases

47. B) PRODUCTION OF
ANTIBODIES:
 The in vitro methods for production of mABs
are the methods of choice because of:
 The ease of culture for production.
 Less economic consideration compared with
the use of animals.
 These advantages make the in vitro methods
meet more than 90% of the needs for mABs.

48. PRODUCTION OF ANTIBODIES
CONT…
 The ability to generate hybridomas has been
stimulated the use of the in vitro methods for
mABs production
Practical uses of the in vitro produced mABs:
 Diagnostic tests for the identification of small
quantities of specific antigens.
 ▫mABs also are used therapeutically: OKT3
recognizes a surface antigen (CD3) on T cell
and is one of the most effective agents in
preventing immunological rejection of
transplanted kidneys.

49. PRODUCTION OF ANTIBODIES
CONT…
 Various mAbs designed to destruct tumor
cells by targeting a membrane bound
protein antigens specifically expressed by
these cells.
 The conjugation of radio active or toxic
compounds to the antibody can result in a
localized high concentration resulting in
cytotoxicity to the target cells.

50. C) RECOMBINANT PROTEINS:
 This idea based on the ability to transfect cells
with isolated genes and amplify it to allow
high level of expression of the corresponding
proteins.
 Proteins extracted from biological sources
have been important for the substitution
therapy since the 1920s when Best and
Banting used insulin to treat diabetes.

51. RECOMBINANT INSULIN
 Eli Lilly Company received approval to market human
insulin under the trade name Humulin in 1986.
 Wockhardt Limited has launched India's first
recombinant human insulin product called Wosulin
in 2003.
 fourth company in the world - and the first outside
US and Europe .
 The single-most advantage of using recombinant
human insulin is that it has identical amino acid
sequence as that of naturally produced insulin in the
human body.

52. SOME EXAMPLES FOR THESE
BIOLOGICAL PRODUCTS:
 1- Interferone:
 Discovered when Isaacs and Lindenmann
(1957) found that culture medium taken from
cells that had supported viral growth could
protect non-infected cells from a subsequent
viral infection.
 2- Tissue plasminogen activator (TPA):
 TPA was produced in large scale by Genenteck
from transfected CHO-K1 cells. It is used to
prevent undesirable formation of fibrin clots
in the bloodstream.

53. SOME EXAMPLES FOR THESE
BIOLOGICAL PRODUCTS:
CONT…
 3- Blood clotting factors:
 For example, factor VIII is produced in
large scale by Bayer through transfection of
the mammalian kidney cell line (BHK) with an
appropriate gene.

54. TISSUE ENGINEERING
 This means the re-constitution of human
tissues from the combinations of cell types
grown in culture. This is an important
prospect for future therapeutic treatment
with organ failure. This include:
 1- Artificial tissues:
 The re-constitution of skin following severe
burns is considered the most successful
application of tissue engineering.
 Artificial skin can be formed from two layers
derived from cultured human cells:

55. TISSUE ENGINEERING
CONT…
 ▫ A dermal-equivalent formed from fibroblasts.
 ▫ An epidermal-equivalent which is layered on the
dermal surface.
 2- Artificial organs:
 Construction of organs in in vitro have met technical
difficulties:
 ▫ Multiple cell types require complex scaffolds and an
extracellular matrix to support the functional
relationship between cells.
 ▫ Multiple cell layer require a nutrient supply
equivalent to blood capillaries in vivo.

56. CELL THERAPY
 Literally, cell therapy means treatment with cells, i.e.
replacing diseased or dysfunctional cells with healthy
functioning ones.
 For example:
 • When hematopoietic cells are vulnerable to
destruction by any cytotoxic drugs used in
chemotherapy to eradicate residual tumor cells.
 • Bone marrow pluripotent stem cells can be isolated
and expanded prior to chemotherapy to provide a
source of mature hematopoeitic cells following
chemotherapy.

57. GENE THERAPY
 The concept of gene therapy is that a missing
or faulty gene is replaced by a normal
working gene.
 The process involves the transfection of a
specific gene into cells of patient with an
identified and well characterized genetic
disease.
 The gene can be introduced into inside the
patient (in vivo) or outside the patient (ex
vivo).

58. GENE THERAPY
CONT…
 For example: severe combined immunodeficiency
(SCID) is associated with a defective copy of a gene,
required for the expression of the enzyme adenosine
deaminase (ADA).
 Treatment by gene therapy involves:
 1. Isolation of bone marrow stem cells from the patient.
 2.Infection of the cells with a retrovirus constructed to
carry the ADA gene.
 3. The transfected stem cells are then introduced into
the bone marrow of the patient where they can
proliferate and differentiate into immunocompetent
cell.

59. Thanks

60. WHAT IS NEEDED?
 Tissue culture, of animal has several
critical requirements:
 Appropriate tissue (some tissues culture
better than others)
 Aseptic (sterile) conditions, as
microorganisms grow much more quickly
than animal tissue and can over run a
culture

61. WHAT IS NEEDED, II
 A suitable growth medium containing
energy sources and inorganic salts to
supply cell growth needs. This can be
liquid or semisolid
 Growth regulators -. In animals, the
growth substances are provided in serum
from the cell types of interest.
 Frequent subculturing to ensure adequate
nutrition and to avoid the build up of waste
metabolites

62. LIMITATIONS
Category
Necessary
Expertise
Environmental
control
Quantity
and cost
Examples
 Sterile handling
 Chemical contamination
 Microbial Contamination
 Cross-contamination
 Workspace
 Incubation, pH control
 Containment and disposal of biohazards
 Capital equipment for scale-up
 Medium, serum
 Disposable plastics 8/5/2022
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63. LIMITATIONS
Category
Genetic
instability
Phenotypic
instability
Identification
of cell type
Examples
 Heterogeneity, variability
 Dedifferentiation
 Adaptation
 Selective overgrowth
 Markers not always expressed
 Histology difficult to recreate and atypical
 Geometry and microenvironment change cytology
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64. WHY IS CELL CULTURE USED FOR?
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, process
and triggering of aging & nutritional studies
 Toxicity testing
Study the effects of new drugs
 Cancer research
Study the function of various chemicals,
virus & radiation to convert normal cultured cells
to cancerous cells

65.  Genetic Engineering
transfect the cultured cells with foreign DNA and to
see the expression of these genes, production of
recombinant protein like interferons, human growth
hormone etc
 Gene therapy
Cells having a functional gene can be replaced to
cells which are having non-functional gene
 Karyotyping studies
Helps in early diagnosis of abnormalities. Turner’s
syndrome, Down syndrome etc
Contd….

66. CONTD….
 Replacement of damaged tissue and cells
Tissue grafting
 Virology
Cultivation of virus for vaccine production, also
used to study there infectious cycle
 Production of medicinal & commercial proteins
large scale production of viruses for use in vaccine
production e.g. polio, rabies, chicken pox, hepatitis B &
measles AND monoclonal antibodies, insulin,
hormones etc