unit 10 experimental pharmacology PC_530

2. HISTORY OF CELL CULTURE
The plaque assay remains to be the
most widely used technique for virus
isolation and purification, and to
determine viral titers. The basis of the
technique is to measure the ability of a
single infectious virus to form a
“plaque” on a confluent monolayer
culture of cells.
A fibroblast is the most
common type of cell found
in connective tissue.
Fibroblasts secrete collagen
proteins
HT-3 is a human cervical
carcinoma cell line that grows
in adherent culture.

3. Major developments in cell
culture technology

5. What is Tissue/Cell Culture?
Refers to the removal of cells or tissues or organs from an animal or plant and
their subsequent placement in a favorable artificial environment conducive
to growth
Environment usually consists of a suitable glass or plastic culture vessel
containing a liquid or semisolid medium that supplies the nutrients essential
for survival and growth.

6. Primary Culture
Primary culture refers to the stage of the culture after the cells are
isolated from the tissue and proliferated under the appropriate
conditions until they occupy all of the available substrate (i.e., reach
confluence).
Ex-plant culture Enzymatic dissociation

7.  Cell Line- a defined population of cells that can be maintained in
culture for an extended period of time, retaining stability of
certain phenotypes and functions. Cell lines are usually clonal,
meaning that the entire population originated from a single
common ancestor cell.
 After the first subculture, the primary culture becomes known as a
cell line or sub clone. Cell lines derived from primary cultures have a
limited life span (i.e., they are finite) and as they are passaged, cells
with the highest growth capacity predominate, resulting in a degree of
genotypic and phenotypic uniformity in the population.
 Cell Strain- If a subpopulation of a cell line is positively selected
from the culture by cloning or some other method, this cell line
becomes a cell strain
Buying and Borrowing- Buy established cultures from
organizations such as ATCC or Coriell institute of Medical
Research (ccr.coriell.org) or RIKEN cell bank or National Centre
for Cellular Sciences (NCCS)

8. Cell lines can be
propagated as two types of
cultures
Suspension culture.
Anchorage independent
Cell cultures from
hematopoietic cells.
Ex: Leukemia cells,
Multiple melanoma cells.
Cell cultures from organ or
tissues.
Ex: Epithelial cells,
Fibroblasts.
Monolayer culture.
Anchorage dependent

9. Types of Cells
Cultured cells are usually described based on their
morphology (shape and appearance) or their functional
characteristics.
There are three basic morphologies:
1. Epithelial-like: cells that are attached to a substrate
and appear flattened and polygonal in shape.
2. Lymphoblast-like: cells that do not attach normally to a
substrate but remain in suspension with a spherical
shape.
3. Fibroblast-like: cells that are attached to a substrate
and appear elongated and bipolar, frequently forming
swirls in heavy cultures
Epithelial like
Lymphoblast like
Fibroblast like

10.  The artificial environment created in the laboratory is generally
known as media.
 A media comprises an appropriate source of energy for the cells
which they can easily utilize and compounds which regulate the
cell cycle.
 The choice of media is cell type specific and often empirical and
there is no “all purpose” medium.
 It should provide many nutrients, buffering capacity, isotonic,
and should be sterile.
 Characteristics and compositions of the cell culture media vary
depending on the particular cellular requirements as shown in
Table 1

11. Most animal cell culture media are generally having following 10 basic
components and they are as follows:
1. Energy sources: Glucose, Fructose, Amino acids
2. Nitrogen sources: Amino acids
3. Vitamins: Generally water soluble vitamins B & C
4. Inorganic salts: Na+, K+, Ca2+, Mg2+
5. Fat and Fat soluble components: Fatty acids, cholesterols
6. Nucleic acid precursors
7. Antibiotics
8. Growth factors and hormones
9. pH and buffering systems
10. Oxygen and CO2 concentration.

12. Media used for tissue culture may be grouped into two broad categories:
1. Natural media
2. Artificial media.
The choice of medium depends mainly on the type of cells to be cultured
(normal, immortalized or transformed), and the objective of culture (growth,
survival, differentiation, production of desired proteins).
Non transformed or normal cells (finite life span) and primary cultures from
healthy tissues require defined quantities of proteins, growth factors and
hormones.
But immortalized cells (spontaneously or by transfection with viral sequences)
produce most of these factors, but may still need some of the growth factors
present in the serum.
In contrast, transformed cells (autonomous growth control and malignant
properties) synthesize their own growth factors; in fact, addition of growth
factors may even be detrimental in such cases. But even these cultures may
require factors like insulin, transferrin, silenite, lipids, etc.
Types of Media

13. 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
3. Tissue extracts.
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
4. Specialized functions.

14. The various artificial media developed for cell cultures may be
grouped into the following four classes:
(i) Serum containing media
(ii) Serum free media
(iii) Chemically defined media
(iv) Protein free media.
SERUM
Liquid yellowish, clear content left over after fibrin and cells are
removed from the blood is known as serum.
Calf (bovine), foetal bovine, or horse are used, in some cases human.
Fetal bovine serum (FBS) (10-20% v/v) is the most commonly applied
supplement in animal cell culture media.

15. Contents of Serum

17. Functions of Serum in the Culture Medium
It provides the basic nutrients for cells; the nutrients are present both in the
solution as well as are bound to the proteins.
It provides several hormones, e.g., insulin, which is essential for growth of nearly
all cells in culture, cortisone, testosterone, prostaglandin, etc.
It contains several growth factors, e.g., platelet derived growth factor (PDGF),
transforming growth factor β (TGF-β), epidermal growth factor, etc.; these are
present in concentrations of μg/l.
A major role of serum is to supply proteins, e.g., fibronectin, 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.

18. It provides several binding proteins, e.g., albumin, transferrin, etc., which
carry other molecules into the cell. For example, albumin carries into cells
lipids, vitamins, hormones, etc. Transferrin usually carries Fe in a nonbasic
form, but binding of transferrin to its receptor in cell membrane is believed
to be mitogenic.
 It increases the viscosity of medium and thereby, protects cells from
mechanical damages, e.g., shear forces during agitation of suspension
cultures.
 Protease inhibitors present in the serum protect cells, especially
trypsinised cells, from proteolysis.
The serum also provides minerals, like Na+, K+, Fe2+, Zn2+, etc.
 It also acts as a buffer.

19.  The chemical composition of these supplements may vary between
lots, even from a single manufacturer.
 The supplements of animal or human origin may also be
contaminated with infectious agents (e.g., mycoplasma and
viruses) which can seriously undermine the health of the cultured
cells when these contaminated supplements are used in cell culture
media formulations and may pose a health risk in cell therapy and
other clinical applications.
 A major fear is the presence of prions causing spongiform
encephalopathy in humans or animals.
 Cell surface chemistry, which is a critical portion of the in vitro
microenvironment for many cell types, can be adversely modified
via adsorption or incorporation of serum or extract proteins.
Limitation of Media with Serum

20. The use of undefined components such as serum or animal extracts
also prevents the true definition and elucidation of the nutritional and
hormonal requirements of the cultured cells, thus eliminating the
ability to study, in a controlled way, the effect of specific growth
factors or nutrients on cell growth and differentiation in culture.
Undefined supplements prevent the researcher from studying
aberrant growth and differentiation and the disease related changes in
cultured cells.
Using cell culture media in the industrial production of biological
substances, serum and animal extract supplementation of culture
media can also complicate and increase the costs of the purification of
the desired substances from the culture media due to nonspecific co-
purification of serum or extract proteins.

21. SERUM-FREE MEDIA
 Media, which often are specifically formulated to support the culture of a
single cell type, incorporate defined quantities of purified growth factors,
lipoproteins and other proteins usually provided by the serum or extract
supplement.
 Since the components (and concentrations thereof) in such culture media are
precisely known, these media are generally referred to as “defined
culture media” and often as “serum-free media” or “SFM.”
 Use of SFM facilitates the investigation of the effects of a specific growth
factor or other medium component on cellular physiology, which may be
masked when the cells are cultivated in serum- or extract-containing Media
 SFM typically contain much lower quantities of protein (indeed, SFM are
often termed “low protein media”) than those containing serum or extracts,
rendering purification of biological substances produced by cells cultured in
SFM far simpler and more cost-effective

22. MEDIUM APPLICATION AUTHORS
Basal medium Growing cells with serum Eagle 1965
Minimal essential
medium (MEM)
Growing cells with dialyzed
serum
Eagle 1959
Dulbecco’s modified
Eagle’s medium
Virus transfected cells, high
density growth with serum
Dulbecco and
Freeman 1959
Ham’s F10 medium Chick embryo cells, serum Ham 1963
RPMI 1640 Human leukemic cells and
Hybridomas
Moore and Kitamura
1968
Mc Coy’s5A medium Human lymphocytes Mc Coy et.al 1959
Neuroblast medium CNS Neurons Breweret al 1994
MCDB 131 Human endothelium Knedler and Ham
1987
Medium199 Chick embryo fibroblasts Morgal et.al 1950
Protein Free Media
DMEM (Dulbecco's Modified Eagle Medium) Adherent culture.
RPMI (Roswell Park Memorial Institute medium ) Suspension culture.

23. PHYSICOCHEMICAL PROPERTIES OF CULTURE MEDIUM
1. pH :Most cell lines grow well at pH 7.4.
•Transformed cells may do better at ph 7.0–7.4.
• Phenol red is commonly used as an indicator. It shows-
•More pink at pH 7.6,
•Purple at pH 7.8
•Red at pH 7.4,
•Orange at pH 7.0,
•Yellow at pH 6.5,
•Lemon yellow below pH 6.5.
2. CARBON DIOXIDE :
•To maintain buffering capacity of the medium and to compensate the lost
dissolved gas from medium.
•Generally 0-10% CO2 is supplied.
•5% gas supply from cylinders is widely used.
•CO2 levels are measured by FYRITE UNIT.
DISADVANTAGE
Phenol red has weak
estrogenic activity.

24. 3. BUFFERING :
It is required under two conditions.
(1) open dishes, wherein the evolution of co2 causes the pH to rise
and
(2) overproduction of CO2 and lactic acid in transformed cell lines at
high cell concentrations, when the pH will fall.
Widely used buffers:
1.Bicarbonate buffer.
2.Tris buffer.
3.Zwitter ionic buffer (HEPES).
(N-2-Hydroxy ethyl piperazine-N-2-ethane sulfonicacid)
Low Toxicity, Low Cost, and
Nutritionally Benefit.
4. OSMOLALITY
* Osmolalities between 260 mosmol/kg and 320 mosmol/kg are quite
acceptable for most cells but, once selected, should be kept consistent
at ±10 mosmol/kg.
*Slightly hypotonic medium may be better for petri dish or open-plate
culture to
compensate for evaporation during incubation.
Modifiers : NaCl, Dextrose

25. 5. OXYGEN :
As there is no oxygen carrier(hb)cells rely chiefly on dissolved O2, which can be
toxic due to the elevation in the level of free radicals. Providing the correct O2
tension is therefore, always a compromise between fulfilling the respiratory
requirement and avoiding toxicity.
So free radical scavengers, such as
Glutathione,
2-Mercaptoethanol (β-mercaptoethanol),
or Dithiothreitol ,
are added into the medium.
SELENIUM is incorporated into the medium which is cofactor for
GLUTATHIONE thus avoid toxicity.
6. TEMPERATURE
●The temperature recommended for most human and warm-blooded animal cell
lines is 37◦C, close to body heat.
●Cultured mammalian cells will tolerate considerable drops in temperature, can
survive several days at 4◦c, and can be frozen and cooled to −196◦c.

26. Finite v/s Continuous Cell Line
Normal cells usually divide only a limited number of times before losing
their ability to proliferate, which is a genetically determined event known
as senescence, these cell lines are known as finite.
However, some cell lines become immortal through a process called
transformation, which can occur spontaneously or can be chemically or
virally induced. When a finite cell line undergoes transformation and
acquires the ability to divide indefinitely, it becomes a continuous cell
line.

27. MEDIUM APPLICATION AUTHORS
Basal medium Growing cells with serum Eagle 1965
Minimal essential
medium (MEM)
Growing cells with dialyzed
serum
Eagle 1959
Dulbecco’s modified
Eagle’s medium
Virus transfected cells, high
density growth with serum
Dulbecco and
Freeman 1959
Ham’s F10 medium Chick embryo cells, serum Ham 1963
RPMI 1640 Human leukemic cells and
Hybridomas
Moore and Kitamura
1968
Mc Coy’s5A medium Human lymphocytes Mc Coy et.al 1959
Neuroblast medium CNS Neurons Breweret al 1994
MCDB 131 Human endothelium Knedler and Ham
1987
Medium199 Chick embryo fibroblasts Morgal et.al 1950
SOME COMMON COMMERCIALLYAVAILABLE MEDIA
DMEM (Dulbecco's Modified Eagle Medium) Adherent culture.
RPMI (Roswell Park Memorial Institute medium ) Suspension culture.

28. PHYSICOCHEMICAL PROPERTIES OF CULTURE MEDIUM
1. pH :Most cell lines grow well at pH 7.4.
•Transformed cells may do better at ph 7.0–7.4.
• Phenol red is commonly used as an indicator. It shows-
•More pink at pH 7.6,
•Purple at pH 7.8
•Red at pH 7.4,
•Orange at pH 7.0,
•Yellow at pH 6.5,
•Lemon yellow below pH 6.5.
2. CARBON DIOXIDE :
•To maintain buffering capacity of the medium and to compensate the lost
dissolved gas from medium.
•Generally 0-10% CO2 is supplied.
•5% gas supply from cylinders is widely used.
•CO2 levels are measured by FYRITE UNIT.
DISADVANTAGE
Phenol red has weak
estrogenic activity.

29. 3. BUFFERING :
It is required under two conditions.
(1) open dishes, wherein the evolution of co2 causes the pH to rise and
(2) overproduction of CO2 and lactic acid in transformed cell lines at high cell
concentrations, when the pH will fall.
Widely used buffers:
1.Bicarbonate buffer.
2.Tris buffer.
3.Zwitter ionic buffer (HEPES).
(N-2-Hydroxy ethyl piperazine-N-2-ethane sulfonicacid)
Low Toxicity, Low Cost, and
Nutritionally Benefit.
4. OSMOLALITY
* Osmolalities between 260 mosmol/kg and 320 mosmol/kg are quite acceptable
for most cells but, once selected, should be kept consistent at ±10 mosmol/kg.
*Slightly hypotonic medium may be better for petri dish or open-plate culture to
compensate for evaporation during incubation.
Modifiers : NaCl, Dextrose

30. properties finite continuous
Finite versus Continuous cell lines:

32. CATEGORY ADVANTAGES
ADVANTAGES OF TISSUE CULTURE

33. DISADVANTAGES OF TISSUE CULTURE
CATEGORY EXAMPLES

34. TYPES OF TISSUE CULTURE
 ORGAN CULTURE: Three dimensional culture of undisagregated tissue
retaining some or all of the histological features of tissue in vivo.
 CELL CULTURE: Culture derived from dispersed cells taken from the
original tissue, dispersed by enzymatic, mechanical or chemical dis
aggregation. The source of cells can also be from primary cultures already
developed else where.
 HISTOTYPIC CULTURE: Reaggregation of cells into a three dimensional
tissue like structure by cultivation of high density cells in artificial media or
matrix (e.g.Collagen gel).
 ORGANOTYPIC CULTURE: Recombining cells of different lineages into
organ like structures.

35. BIOLOGY OF CULTURED CELLS
Good environment of culturing cells is based on:
1.Nature of substrate.
2.Degree of contact with other cells.
3.Constitution of medium.
4.Constitution of gas.
5.Incubation temperature.

36. Cell–cell adhesion molecules
Cell–substrate interactions
Cell Adhesion Molecules (CAMs) are proteins located on the cell surface
involved with the binding with other cells or with the extracellular
matrix(ECM) in the process called cell adhesion.
INTEGRINS: Are the receptors for
matrix molecules such as fibronectin,
entactin, laminin and collagen which bind
to them via a specific motif usually
containing the arginine–glycine–aspartic
acid (RGD) Sequence.
* Each integrin comprises one α and one
β subunit, the extracellular domains of
which are highly polymorphic, thus
generating considerable diversity among
the integrin.
These proteins are self-interactive;
that is, homologous molecules in
opposing cells interact with each
other
 E-cadherin------ Epithelia cell
 N-cadherin------ Nerve cell
 P-cadherin-------Placenta
Selectins: which binds to the fucosylated receptors
CD34,
GlyCAM-1(glycosylation dependent cell adhesion molecule-1),
PSGL-1(P-selectin glycoprotein ligand-1).
E-Selectin
L-Selectin

37. CADHERINS INTEGRINS

38. Cell Adhesion

39. Evolution of a Cell Line
Lag Phase: The time
the cell population
takes to recover from
sub culture, attach to
the culture vessel
Log Phase: cell
number begins to
increase exponentially.
Plateau Phase: growth
rate slows or stops due
to exhaustion of
growth medium or
confluency

40. Preparation of primary culture

41. Adherent Suspension Culture
There are two basic systems for growing cells in culture, as
monolayers on an artificial substrate (i.e., adherent culture) or
free-floating in the culture medium (suspension culture).

42. REPLACEMENT OF MEDIUM OR FEEDING
A Drop in pH
The rate of fall and absolute level should be considered. Most cells stop
growing as the pH falls from pH 7.0 to pH 6.5 and start to lose viability
between pH 6.5 and pH 6.0, so if the medium goes from red through
orange to yellow, the medium should be changed.
Cell Concentration
Cell Type
Morphological Deterioration.

43. Sub-culturing or passaging
Sub culturing is the removal of the medium and transfer of cells from a
previous culture into fresh growth medium, a procedure that enables the
further propagation of the cell line or cell strain.
When to Subculture?
Density of Culture
Time Since Last Subculture
Exhaustion of Medium.
Requirements for Other Procedures

44. Sub-culturing of monolayer

45. Sub-culturing of suspension culture

46. (a) Bacteria.
(b) Yeast.
(c) Mold.
(d) Mycoplasma
Types of Contamination

47. Characteristic features of microbial contamination are as
follows:
(1) A sudden change in pH, usually a decrease with most bacterial infections
(Plate 16a), very little change with yeast until the contamination is heavy, and
sometimes an increase in pH with fungal contamination.
(2) Cloudiness in the medium , sometimes with a slight film or scum on the
surface or spots on the growth surface that dissipate when the flask is moved.
(3) Under a low-power microscope (∼×100), spaces between cells will appear
granular and may shimmer with bacterial contamination.
(4) Under high-power microscopy (∼×400), it may be possible to resolve
individual bacteria and distinguish between rods and cocci.

48. ROUTES TO CONTAMINATION
Technique manipulations, pipetting, dispensing, etc.
Work Operator hair, hands, breath, clothing surface.
Materials and reagents Solutions.
Glassware and screw caps.
Culture flasks and media bottles in use.
Equipment and Facilities Room air. Laminar-Flow Hoods.
CO2, humidified incubators.
Importation of Biological Materials Tissue samples.
Incoming cell lines.

49. Cell lines in continuous culture are prone to variation due to selection in early-passage
culture senescence in finite cell lines and genetic and phenotypic instability in
continuous cell lines.
Why cryopreservation has to be done?
Reasons are as follows:
(1) Genotypic drift due to genetic instability.
(2) Senescence and the resultant extinction of the cell line.
(3) Transformation of growth characteristics and acquisition.
of malignancy-associated properties.
(4) Phenotypic instability due to selection and dedifferentiation.
(5) Contamination by microorganisms .
(6) Cross-contamination by other cell lines.
(7) Misidentification due to careless handling.
(8) Incubator failure.
(9) Saving time and materials maintaining lines not in immediate use.
(10) Need for distribution to other users.
Cryopreservation

50. PRINCIPLES OF CRYOPRESERVATION
Optimal freezing of cells for maximum viable recovery
on thawing. This is achieved
(a) by freezing slowly to allow water to leave the cell but not so slowly that
ice crystal growth is encouraged,
(b) by using a hydrophilic cryoprotectant to sequester water,
(c) by storing the cells at the lowest possible temperature to
minimize the effects of high salt concentrations on protein denaturation in
micelles within the ice, and
(d) by thawing rapidly to minimize ice crystal growth and generation of
solute gradients formed as the residual intracellular ice melts.
Cell Concentration
Normally at 1 × 105/mL, 1 × 107 should
be frozen in 1 mL of medium, and, after thawing the cells, the whole 1 mL
should be diluted to 20 mL of medium,giving 5 × 105 cells/mL .

51. Freezing Medium
Dimethyl sulphoxide (DMSO)- widely
used.
Glycerol,
Polyvinyl pyrrolidine (PVP),
Polyethylene glycol (PEG),
Hydroxy Ethyl Starch (HES),
Trehalose.
DMSO : drawbacks
Should be colorless.
Stored in glass containers if not it will leach
impurities from plastic or rubber.
Should be diluted if not toxic to cells.
Glycerol: On long storage toxic to cell lines.
Widely used freezing mixture
40%v/v medium with 10%serum,
40%v/v FCS,
20%V/V DMSO/Glycerol.

52. Procedure for freezing cells

53. Thawing Cells

55. Medium-Sized Tissue Culture Laboratory layout

56. Medium-Sized Tissue Culture Laboratory layout

57. Layout for open work bench in laminar airflow hood

58. EQUIPMENT AND MATERIALS REQUIRED
ASEPTIC AREA:
Laminar flow hoods,
Inverted microscope,
Centrifuge,
Waterbath,
Refrigerator,
Freezer (-200 C, -800 C),
Liquid nitrogen freezers,
Trolleys and carts,
INCUBATORS:
Normal incubator,
Humid CO2 Incubator,
Hemocytometer slides,
Cell counter.
WASH UPAREA:
Washing sink,
Pipette washer,
Pipette drier,
Glassware washer,
MEDIA
PREPARATION:
Millipore water purifier,
Suction pump,
Conductivity meter,
Magnetic stirrers,
Digital balance,
PH meter,
Osmometer.
STERILIZATION
EQUIPMENT:
Autoclave,
 Hot air oven,
Membrane filters.
CONSUMABLES:
Pipettes,
Culture flasks,
Petri dishes,
 Multiwell plates,
Tip and tipboxes,
Centrifuge tubes,
Glass bottles and
measuring cylinders.

59. Cell Culture Hoods
unidirectional flow of HEPA-filtered air over the work area.
HORIZONTAL
*Blowing parallel to the work
surface.
* Provides protection to the
culture (if the air flowing towards
the user) or to the user (if the air
is drawn in through the front of
the cabinet by negative air
pressure inside).
VERTICAL
*Blowing from the top of the
cabinet onto the work surface.
* Vertical flow hoods also provide
significant protection to the user
and the cell culture.

60. HORIZONTAL
VERTICAL

61. *Dry incubators are more
economical but require the cell
cultures to be incubated in sealed
flasks to prevent evaporation.
* Placing a water dish in a dry
incubator can provide some
humidity, but they do not allow
precise control of atmospheric
conditions in the incubator.
*Humid CO2 incubators are more
expensive, but allow superior control of
culture conditions.
*They can be used to incubate cells
cultured in Petri dishes or multi-well
plates, which require a controlled
atmosphere of high humidity and
increased CO2 tension.
DRY INCUBATOR HUMID CO2 INCUBATOR

62. STERILIZATION EQUIPMENT:
FREESTANDING AUTOCLAVE HOT AIR OVEN

64. SPECIALIZED EQUIPMENT
Flow cytometer
RT PCR
Colony counter

65. Culture vessels and their characteristics

67. SUBSTRATES
ATTACHMENT FOR THE GROWTH AND SPREADING
Cells shown to require attachment
for growth are said to be
‘anchorage dependent’.
cells that have undergone transformation
and can grow in suspension are said to be
‘anchorage independent’.
SUBSTRATE MATERIALS
Disposable plastic:
1.Single-use sterile polystyrene flasks:
●They are usually of good optical quality.
●The growth surface is flat,
●Provides uniformly distributed and reproducible monolayer
cultures. As manufactured, polystyrene is hydrophobic and does not
provide a suitable surface for cell attachment, so tissue culture
plastics are treated by corona discharge, gas plasma, or γ -
irradiation, or chemically, to produce a charged, wettable surface.

68. 2.Polytetrafluorethylene (PTFE; Teflon): Two forms
charged (hydrophilic) uncharged (hydrophobic)
monolayer cells,
organotypic culture.
Suspension cultures,
macrophages.
Others:
polyvinylchloride (PVC),
polycarbonate,
Melinex,
Thermanox (TPX),
Polymethyl methacrylate.
Fibres :
Rayon,
Nylon,
Poly-l-lactic acid (PLA),
Polyglycolic acid.
Derivatized substrates with:
RGD tripeptide,
N-Hydroxy sulphosuccimide etc.

69. TREATED SURFACES
1.Extracellular matrix (ECM)
Some cell lines like 3T3 or MRC-5 cells (mouse & human fibroblasts
respectively)
produce ECM.
When flask is washed with triton x-100 the ecm will be remained.
These flasks can be used for culturing cell lines of our interest.
2.Substrate coating:
Treat the surface with collagen, fibronectin, laminin.
3.Commercially available matrices: Matrigel, Natrix, Laminin.
Suspension cultures:
Attachment is not required but uniform distribution of cell lines must be there
i.e. no sedimentation should occur. So suspending agents are used.
Ex : Agar, Agarose, Methocel

70. GOOD ASEPTIC TECHNIQUES:

71. GOOD ASEPTIC TECHNIQUES:

72. METHODS OF STERILIZATION

73. Media should provide many nutrients, buffering capacity, isotonic and
sterile. Main components are:
 Energy sources: Glucose, Fructose, Amino acids.
 Nitrogen sources: Amino acids.
 Vitamins: Water soluble vitamin B & C.
 Inorganic salts: Na+, K+, Ca2+, Mg2+ .
 Fat and Fat soluble components: Fatty acids, Cholesterols.
 Nucleic acid precursors.
 Antibiotics.
 Growth factors and hormones.
 pH and buffering systems.
 Oxygen and CO2 concentration.
COMPONENTS OF MEDIUM
MEDIA SUPPLEMENTS:
1.L-Glutamine 200mM,
2.Fetal Bovine serum 10%v/v,
3.HEPES- 25mM,
4.Antibiotics.

74. ROLE OF MEDIUM
Nutrient support
Maintenance of pH and osmolality
Buffering system
Growth factors
Metabolic support
Antioxidants and reducing agents
Most media (MEM and DMEM) were developed with serum
supplementation
Some are tailored without the serum support to study the
specific effects of defined growth factors
“Special Use Media” are now commercially available for
specific cell lines and contain undisclosed components

75. The three basic classes of
media are:
Basal media,
Reduced-serum media,
Serum-free media.
Differ in their requirement for
supplementation with serum.
BASAL MEDIA
The majority of cell lines grow well in basal media, which contain amino acids,
vitamins, inorganic salts, and a carbon source such as glucose, but these basal
media formulations must be further supplemented with serum.
REDUCED-SERUM MEDIA
Another strategy to reduce the undesired effects of serum in cell culture
experiments is to use reduced-serum media. Reduced-serum media are basal media
formulations enriched with nutrients and animal-derived factors, which reduce the
amount of serum that is needed.
SERUM-FREE MEDIA:
Replacing the serum with appropriate nutritional and hormonal formulations.
TYPES OF MEDIUM

76. CONSTITUENTS OF SERUM
Proteins and Polypeptides
Albumin
Fetuinb
Fibronectin
Globulins
Protease inhibitors: α1-antitrypsin,
α2-macroglobulin.
Transferrin.
Growth factors:
EGF, PDGF, IGF-I and -II,
FGF, IL-1, IL-6.
Amino acids
Lipids
Cholesterol
Fatty acids
Linoleic acid
Phospholipids.
Hormones:
Hydrocortisone
Insulin
Triiodothyronine
Thyroxin.
Vitamins:
Vitamin A
Folate.
Polyamines:
Putrescine,
spermidine.
Urea
Inorganics:
Calcium
Chlorides
Iron
Potassium
Phosphate
Selenium
Sodium
Zinc.
Carbohydrates
Glucose
Hexosamine
Lactic acid
Pyruvic acid

77. DISADVANTAGES OF SERUM
1.Physiological Variability:
Reason : components include nutrients (amino acids, nucleosides, sugars,
etc.),peptide growth factors, hormones, minerals and lipids, the concentrations
and actions of which have not been fully determined.
2.Shelf Life and Consistency:
Serum deteriorating during the storage time. So It must be replaced with another
batch that may be selected as similar, but will never be identical, to the first
batch..
3.Quality Control: Extensive testing to ensure that the replacement is as close
as possible to the previous batch.
4.Specificity:
If more than one cell type is used, each type may require a different batch of
serum, so that several batches must be held on reserve simultaneously.
5.Availability:
the supply of serum is restricted now-a-days because of drought in the cattle-
rearing areas, the spread of disease among the cattle, or economic or political
reasons.

78. 6.Downstream Processing: The presence of serum creates a major obstacle to
product purification
7.Contamination: Serum is frequently contaminated with viruses,
8.Cost: serum constitutes the major part of the cost of a bottle of medium
(more than 10 times the cost of the chemical constituents)
9.Growth Inhibitors: As well as its growth-promoting
activity, serum contains growth-inhibiting activity, and although stimulation
usually predominates, the net effect of the serum is an unpredictable
combination of both inhibition and stimulation of growth.
10.Standardization: Standardization of experimental and production
protocols is difficult, both at different times and among different laboratories,
because of batch-to batch variations in serum.
All of the above problems can be eliminated by removal of serum and
specific media with required factors can be used, thus there will be
control over the proliferation and differentiation.

79. DISADVANTAGES OF SERUM-FREE MEDIA:
Multiplicity of Media
Each cell type appears to require a different recipe tumors may vary in
requirements from tumor to tumor, even within one class of tumors. It presents a
problem for laboratories maintaining cell lines of several different origins.
Selectivity
 Some media may select a sub lineage and even in continuous cell lines, some
degree of selection is required.
 Cells at different stages of development (e.g. stem cells vs. committed precursor
cells) may require different formulations, particularly in the growth factor and
cytokine components.
Expensive:
 As the removal of serum also removes the protective, detoxifying action so,high
degree of aseptic conditions must be maintained.
Purity of the reagents and water must be high.
Cell Proliferation: Growth is often slower in serum-free media.
Availability: Although improving steadily, the availability of properly quality-
controlled serum-free media is limited.

80. STERILE FILTRATION
Filtration through 0.1- to 0.2-μm micro porous filters is the method of choice for
sterilizing heat-labile solutions

81. Cell counting is required
» To monitor cells during cell cultures
» For cell preparation or any cell experiment
» To standardize cell samples for analysis.
» Cell proliferation
» Cytotoxicity assays
Cell Viability studies

82. Different Methods to determine cell Viability
http://www.dojindo.com/Protocol/Cell_Proliferation_Protocol.pdf

83. Thymidine/BRDU incorporation assay

84. MTT assay
Factors effect MTT assay
Growing crystals may suggest that marginally soluble formazan
accumulates where seed crystals have begun to deposit
Toxicity of the MTT compound
Interfernce by ascorbic acid, or sulfhydryl-containing compounds
including reduced glutathione,coenzyme A, and dithiothreitol, can
reduce tetrazolium salts non-enzymatically and lead to increased
absorbance values in assay wells

85. MTS/XTT/WST Assay

86. Resazurin assay

87. Protease Viability Marker Assay
Measure resulting fluorescence using a fluorometer (380–400 nm
Ex/505 nm Em).

88. Calcein –AM assay
Record fluorescence using a 490 nm excitation filter and a 520
emission filter.

89. ATP Assay Concept

90. Selection Guide
http://www.interchim.fr/cat/ViabilityCytoxicityAssays

91. REFERENCES
1. Ian Freshney Culture of Animal Cells A Manual
of Basic Technique, 5th Edition.
2. Handbook for cell culture basics (Gibco).