The document provides an overview of animal cell culture techniques, emphasizing the unique characteristics and requirements of animal cells compared to other cell types. It details the structure of animal cells, methods for establishing cell lines through primary cultures and subcultures, as well as factors affecting cell growth, such as pH, temperature, and osmolality. Additionally, it discusses the roles of feeder cells in culture and the different growth phases of cells, including the implications of transformation and immortalization of cell lines.

1. Animal cells: Basic Concepts
Shubham A. Chinchulkar (Regulatory Affairs)
M.Tech (Pharm.)
National Institute of Pharmaceutical education and
Research (NIPER)
shubhamchinchulkar007@gmail.com

2. Introduction
 Cell Culture - cultivation of dispersed cells taken from an original tissue, a primary culture, or a cell line
 Cell culture techniques allow in vitro propagation of various cell lines including those from insects, humans, mice,
rats, and other mammals
 Animal cells: a. More delicate
b. vulnerable to mechanical damage
c. present lower growth rates
d. require more complex culture media and special substrates
 Rigorous aseptic conditions - animal cells grow more slowly than most usual contaminants, such as bacteria and
fungi

3. Typical structure of an animal cell

4. Animal Cell
Components
Plasma
membrane
Cytoplasm
Endoplasmi
c reticulum
Ribosome
Golgi
complex
Mitochondria Lysosome Peroxisome Nucleus
Separates the
intracellular
components
thickness
around 7.5–
10 nm,
controls the
flux of
molecules
between the
cytosol and
the
extracellular
environment.
Cytoplasm
consists of the
cytosol.
Ectoplasm &
Endoplasm
Structures are
Enveloped by
a membrane
that separates
the
endoplasmic
reticulum
cavities or
cisternae.
Cisternae
also serve
as a storage
area.
Protein synthesis,
Lipid
biosynthesis,
relaxation by
reabsorbing Ca2+
Secrete steroid
hormones
Spherical
cell
structures
enriched
with
ribonucleop
rotein with
a diameter
of 15–20
nm.
Proteins
accumulate
in the
cisternae.
Arrangement
is a stack of
circular
flattened
vesicles
Golgi complex
receives
products from
the
endoplasmic
reticulum
and packs
them in
secretory
vesicles
Enzyme-rich
structures that
catalyze the
oxidation of
organic
nutrients
Generate
adenosine
triphosphate
It containd
DNA, RNA and
ribosomes
It can also
divide during
cell replication
Spherical
organelles,
enveloped by a
membrane and
containing
several
hydrolytic
enzymes that
present maximal
activity at acidic
pH
Reservoirs of
different
enzymes
Spherical
organelles
that are 0.3–
1.5 m
in diameter
Interior is
full of
proteins,
frequently in
crystalline
form
Presence of
various
oxidative
enzymes
Very
complex
structure
Two
membranes
of the
nuclear
envelope
form pores
with a
diameter of
around
90 nm

5. Establishing a Cell Line
 Primary culture – culture up to its first subculture or passage & Cell line – after subculture or passage
 Primary cultures – obtained directly from organ or tissue fragments, following enzymic (trypsin or collagenase and
may be mixed with EDTA) or mechanical disaggregation
 Origin – Animal or Human tissue
Tissue
culture
Cell culture
Primary explant
Organ culture

6. Rat Brain
Enzyme digestion
CollagenTrypsin
Cell culture
Tissue fragment
Finely chopped cells
Organ culture
Tissue fragment
Finely chopped tissues
Primary explant

7. Primary cultures
 Initially heterogeneous
 Cells can be maintained in vitro for only a limited period
 During this period, cells generally maintain the differentiated characteristics of the original tissue from which they
were harvested
 Cells may be subjected to considerable stress
a) Enzymatic dissociation of organ fragments
b) Adherent cells breaks cell–cell or cell–surface interactions
 Dissociated cells generally change their shape, becoming rounded and losing their phenotypic polarity, modifying
protein distribution in cell membrane
 Certainly not all cells survive cell manipulation but those that survive should be able to correct any injuries and adapt to
environmental changes

8.  Culture adaptation is
a) Time-consuming
b) Influenced by culture conditions
 Environmental conditioning = cell conditioning
 Environmental conditioning can be achieved by following methods -
I. Release of growth factors into the medium promote cell adherence and proliferation – environment conditioning – cell
conditioning
II. Using feeder cells - 3T3 fibroblastic cells
Gamma radiation
Mitomicin C
Feeder cells Target cells seeding and growthTreatment
Target cells
Environmental conditioning3T3 fibroblastic cells

9. Feeder
cells
Releasing
growth factors
to the culture
media
Detoxifying the
culture medium
Synthetizing
extracellular
matrix proteins
needed to
control the
cultured cell
growth
Acting as a
substrate for the
attachment of
cells
Role of the feeder cells

10.  Nonreplicating viable and metabolically active feeder cells capable of stable synthesis of ligands or cytokines
needed to support the selective expansion of cultured target cells
 Purified growth factors and also the conditioned medium of other cell lines can be used in some instances to
make fastidious cells independent of the presence of feeder cell layers
 3T3 feeder fibroblasts constitutively produce hyaluronic acid (HA)
 Production of HA rises locally in a variety of processes involving cell motility
 HA becomes hydrated, expanding the extracellular space
 This process mechanically weakens intercellular binding facilitating cell detachment and also provides
hydrated tracks for the migration of detached cells, as observed in wound repair and embryonic
morphogenesis

11.  After successive subcultures of a very heterogeneous primary culture, containing many cell types of the original
tissue, a more homogeneous cell line with a higher growth rate may arise
 After successive subcultures of a very heterogeneous primary culture, containing many cell types of the original
tissue, a more homogeneous cell line with a higher growth rate may arise
 Finite cell lines are generally diploid and maintain some degree of differentiation
 These cell lines die after a limited number of generations, the Hayflick limit, which is usually about 30–50
division cycles depending on the origin of the cells
 Immortalization of cell lines – by oncogene or virus or by chemical treatment
 Transformed cell line - Is a cell line that acquired infinite growth after insertion of viral gene components into
the cell's genome

12. Characteristics
of
Transformed
cell lines
Higher
growth rate
Less
dependency
on blood
serum or
selected
growth
factors;
Increase in
cloning
efficiency
Increase in
heteroploidy
Increase in
heteroploidy
Altered cell
morphology
Characteristics of transformed cell lines
Unlimited cell supply
Maintain few
characteristics of their
original tissue
Advantage
Disadvantage

13.  Periodic change of culture medium is necessary when cells are proliferating even in cultures showing no cell
proliferation
 Intervals between medium changes and subcultures may vary depending on the cell line, growth rate, and
metabolism
 Indication of need for culture medium replacement –
a. Increase in cell density
b. pH decrease
c. Nutrient depletion in the medium
d. Alteration in cell morphology
 Animal cells may be anchorage-dependent
 These cells normally proliferate in monolayers and show contact inhibition
 Available surface of culture vessel plays role in cell yield
Cell Line Maintenance

14. MRC-5 Human Lung Fibroblast
Name
Cell type and
tissue origin
Morphology
HeLa Human cervix Epithelial
Vero
African green
monkey kidney
Epithelial
NIH 3T3 Mouse embryo Fibroblast
L929
Mouse connective
tissue
Fibroblast
CHO
Chinese hamster
ovary
Fibroblast
BHK-21
Syrian hamster
kidney
Fibroblast
Adherent Cell Lines
HEK-293 Human kidney Epithelial

15. Suspension Cell Lines
NS0 Mouse myeloma
Lymphoblastoid-
like
Name
Cell type and
tissue origin
Morphology
U937
Human hystiocytic
lymphoma
Lymphoblastoid
Namalwa Human lymphoma Lymphoblastoid
HL60 Human leukemia
Lymphoblastoid-
like
WEHI 231
Mouse B-cell
lymphoma
Lymphoblastoid
YAC 1 Mouse lymphoma Lymphoblastoid
U 266B1 Human myeloma Lymphoblastoid
SH-SY5Y
Human
neuroblastoma
Neuroblast

16.  Blood cells - grow in suspension & Cells derived from solid tissues - adherent cells
Steps involved in adherent cells proliferation –
Adsorption of adhesion
factors to the
substratum
• Step 1
Contact of the cell with
the surface
• Step 2
Cells attach to the
covered surface
producing multivalent
heparin sulphate
proteoglycans, which
bind to cell membrane
glycoproteins
• Step 3
Cell spreading over the
solid surface occurs
•Step 4

17.  Solid substrata with a hydrophilic surface are required, because vertebrate cells have negative charges non-
uniformly distributed over their external membrane surface
 Subculture of adherent cells - removal of culture medium & detachment of cells from the monolayer
 Trypsin plays role in a cell detachment, additionally other proteases such as a pronase, dispase, and collagenase
will be also be used
 EDTA as a chelating agent added to capture the Ca+ ions involved in cell adhesion process
 Gently tapping or hitting the culture flask by hand will be used to removed the weakly bind cell lines
 Trypsin treatment is not required and subculture is faster and less traumatic for the cells
Advantages of suspension cell culture -
 Large cell quantities can be attained without increasing the superficial area of the substratum
 This can rarely be achieved with a monolayer culture
 Cells in different modes (adherent or suspension) behave differently with regard to proliferation, enzymatic
activity, glycolysis, respiration, specialized product synthesis, and many other properties

18. Cell growth phases
 Normal cells in culture show a sigmoid pattern of proliferative activity which reflects culture adaptation, environmental
conditioning, nutrient availability and, for adherent cells, available free adhesion surface
 Cell growth phases –
i. Lag phase,
ii. Exponential or log growth phase,
iii. Stationary or plateau phase, and
iv. Senescence or death phase
 Cell growth can be mathematically represented by
 In lag phase adherent cells may resynthesize the glycocalyx elements lost during trypsinization, bind, and spread on the
substratum
 During spreading, the cytoskeleton reappears and new structural proteins are synthesized
 Inoculum concentration and the point in the growth phase from which cells were taken in the previous culture
Specific cell growth rate
Cell concentration
Culture time

19.  Cells originating from an actively growing culture have a shorter lag phase than those from a quiescent culture
 Cultures initiated at low cell densities - condition the culture medium more slowly - increase the duration of the lag
phase, which is not desirable
 Cell division can reach 90-100% - cells are in best physiological state – ideal for cell function studies
 The cell doubling time -
 Inoculum concentration, cell growth rate, nutrient availability, and accumulation of inhibitory metabolites are the
crucial factors which influences the log phase
 For adherent cells log phase may also occur - When cells cover all the available growth surface, at which point
contact inhibition restricts further growth
 Stationary Phase - Low nutrient concentrations and accumulation of inhibitory metabolites – cell growth rate
reduced

20.  Cell division is equilibrated with cell death - percentage of cells in division is at most 10%
 Stationary phase - relative increase in specialized protein synthesis - change in cell surface composition - charge
modification may occur
 If the culture medium is replenished with fresh – this phase may prolong
 This is not a stable period for most cell lines, and they are susceptible to injuries
 Decline phase – necrosis (result of an irreversible injury and normal homeostasis is lost) and apoptosis (activation of a
biochemical program involving a cascade of cell components, which is internally controlled, requiring energy)
Necrosis
Apoptosis
May result in inflammation No inflammation

21.  Activation of hydrolases – Autodestruction – when lack of lack of nutrients and oxygen, followed by progressive
disorganization and complete disintegration of the cell
 Apoptosis - affects the neighboring cells and may result in inflammation
 During apoptosis comprise caspase activation, mitochondrial membrane permeation, leakage of diverse molecules
from the mitochondria, nuclease activation, cytoskeleton destabilization, externalization of phosphatidylserine to the
outer membrane, and protein interconversion
 Determination of the cell growth profile is important to evaluate the specific characteristics of a cell line
 Because of changes in cell behavior and biochemistry we need to study the growth curves for all the cell line which
will also help us to establishing the most adequate inoculum concentration, prediction of the length of an experiment,
and the most appropriate time intervals for sampling

22. Influence of environmental conditions on animal cell culture
 Cell culture systems - survival and proliferation
 This figure depicts the factor influencing on cell growth
Cell growth
pH Temperature
Osmolality Gas concentration
Available surface substrata State of the cells at inoculation

23. 1. pH
 Most mammalian cell lines proliferate at pH 7.4 and some normal fibroblasts proliferate well at a pH range between
7.4 and 7.7
 Insect cells show better proliferation at lower pH values, from 6.2 to 6.5
 Acid indicator used to monitor the pH variation - phenol red - rose-colored at pH 7.8 - red at pH 7.4, orange at pH 7.0,
and yellow at pH 6.5
 This compound can interfere with the interpretation of experimental data obtained by the use of fluorescence and
absorbance techniques
 Culture medium needs to be buffered to compensate for CO2 and lactic acid derived from glucose metabolism
 Culture medium buffered with CO2 originating from the gaseous phase, in equilibrium with sodium bicarbonate
(NaHCO3) added to the culture medium

24.  An increase in CO2 tends to increase H+ and HCO3 – in reaction (3), consequently increasing medium acidity
 In compensation, the increase in HCO3 – causes NaHCO3 formation through reaction (4), until an equilibrium is
reached at pH 7.4
 Traditionally, culture media are buffered with sodium bicarbonate at a final concentration of 24 mM
 Cells in lag phase – growing at low densities - not produce CO2 in sufficient quantities to maintain the pH at a
optimal value – decrease CO2 content - increase in the culture equilibrium pH
 Hence, control of CO2 concentration allows appropriate maintenance of culture pH
 The gas phase in contact with cell culture in an incubator is usually adjusted to 5–10% of CO2 in volume (90–95%
air)
 Organic buffer - Hepes (N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid) - most effective at the 7.2–7.6 pH
range - this compound is more resistant to rapid alterations in culture conditions
 For insect cells, culture medium is buffered with sodium phosphate, and the use of CO2 or pH indicators is not
required

25. 2. Osmolality
 The optimal osmolality range for mammalian cells in culture is from 260 to 320 mOsm/kg and for insect cells higher
values are optimal, from 340 to 390 mOsm/kg
 Important to verify the osmolality of all culture media after alterations addition of salt solutions, supplements,
pharmaceuticals, and hormones, or large quantities of buffering agents
 Metabolic transformation also responsible for osmolality changes
 The greater the concentration of the substance dissolved, the higher the osmolality, hence osmolality can also increase
due to evaporation
 Culture flasks are generally not sealed so as to allow equilibrium between culture medium and the CO2 –air gas
mixture
 For open cultures in multiple well plates or in Petri dishes slightly hypotonic culture medium can be more adequate
 Relative humidity of culture environment should be near to saturation to avoid large variation in osmolality during
culture

26. 3. Temperature
 It is having influence on cell growth and it affects the solubility of various medium components, especially of gases
such as CO2 and O2, which have low solubility
 Most mammalian cells - 35–37, Insects cell – 26-28, and old-blooded vertebrate cells normally grow well at lower
temperatures
4. Oxygen Supply
 CO2 and O2 are the most important components of gaseous phases and monitoring and controlling is mandatory
 For most cells, oxygen partial pressure conditions slightly below atmospheric pressure are preferred
 Because of that, maintenance of dissolved oxygen concentration at a range from 30 to 60% for mammalian cells is
important
 Oxygen is first component to be limiting in high cell densities due to its having low solubility in aqueous medium

27.  Aeration can be done in direct way or in a isolated vessel surface aeration, gas bubbling, diffusion through
membranes, increasing O2 partial pressure and atmospheric pressure
 In case of static culture – surface aeration – in a closed system - a void volume 10 times larger than the culture
medium volume is necessary for adequate oxygen supply
 In case of agitated culture – surface aeration – oxygen transfer is dependent on agitation rate and impeller
geometry
 Gas bubbling is a simple and efficient way to transfer oxygen, this procedure can cause damage to animal cells due to
bubble formation
 Alternatively, through hydrophobic membranes (silicon tubes), but this method is inconvenient, presents high cost,
and is difficult to scale up, since the tube length for adequate oxygen supply in high capacity reactors needs to be
very long
 An increase in O2 partial pressure also increases oxygen solubility and its diffusion rate at high cell density,
otherwise oxygen toxicity effects could occur

28.  Although oxygen is essential for cell growth, it can be toxic in high concentrations
 For suspension cultures, cell injury can occur due to the disruption of ascending bubbles or of those associated with
the foam accumulated at the surface
 Serum and the surfactant Pluronic F68, have been employed since the 1950s to protect cells from fluid-
mechanical forces
 Serum – good cell protector agent and attributed to the increase in medium viscosity
 Methylcellulose and carboxymethylcellulose as serum substitutes has shown very promising results
 In bioreactors with intense agitation and bubbling aeration, the use of Pluronic1 F68 at 0.1% (v/v) concentration can
result in an efficient cell-protecting effect

29. 5. Composition and nature of the substratum for cell adhesion
 Glass and plastic are the most common materials employed as a solid substratum, which help cell to for adhesion,
spreading, and proliferation
 Glass (borosilicate Surfaces - have a high silica content ) was only used since long hence ‘‘in vitro’’ means ‘‘in glass’’
 Glass is used at small scale and very rare for large scale
 At the end of 1960s plastic material introduced for routine cell culture in a laboratory
 Glass and plastic (Polystyrene) - hydrophobicity and negative charge, were maintained in these materials
 Nowadays, polystyrene is mostly used plastic because of surface characteristics, low cost, transparency
 For demanding cell line surface should be coated with proteins such as poly-lysine, poly-ornithine, or extracellular
matrixderived proteins such as fibronectin, laminin, and collagens
 Microcarriers as substrates is recommended for adherent cell culture on larger scales, large effective area for cell
adhesion, it produced from glass, dextran, agarose, collagen, or modified polymers

30. 6. Cryopreservation and storage of cell lines
 The cells are unstable when they are present in culture state, consequently changes like alterations in their
morphology, function, growth pattern, and karyotype
 When cells are in frozen state then we can preserve it for long period without change in viability and characteristics
 Cells stock also minimizes the risk of losing a culture due to accidents such as contamination by microorganisms or
other cell lines, or due to failure of equipment such as CO2 incubators
 The cells should be in active growth phase with 90% viability and free of contaminants during storage in frozen
condition
 The optimal cryopreservation conditions are different for each cell line
 The cells should be treated with a cryoprotector to support the freezing and thawing processes
 Optimal cell concentration in a suspension of cryoprotector medium generally within the range of 1x106 to 1x107
cells/ml (depends on the cell type and is determined empirically)

31.  Cryoprotectors (concentrations between 5 and 10% (v/v)) such as glycerol or dimethylsulfoxide (DMSO) affect
membrane permeability, allowing water release from the cell interior during cooling
 With cell dehydration, occurring at between –5oC and –15oC, ice crystals are formed around the cells and not
inside them (prevent lysis)
 Cooling velocity is also critical and should be low (about 1oC/min)
 For cell cultivated in serum medium, cryopreservation medium should also contain fetal bovine serum to
protect cells from the stress associated with the freezing and thawing processes
 Serum is added at concentrations above 20%, and can attain 90% of volume of the cryopreservation medium
 However, storage in vapor phase liquid nitrogen (at –140oC to –180oC) is recommended, avoiding possible
contaminations or cell death in case of cryotube rupture
 During cell thawing, the heating rate should be high
 Flasks containing frozen cells are immersed in water at 37oC, and thawing occurs in about 90 seconds

32.  Fresh culture medium should be gradually added to the thawed cell suspension
 For cell line storage, a cell bank is generally established, with initially three to five flasks. One of these flasks is then
thawed and the cell population is expanded to produce a master bank with about 10 to 20 flasks, depending on future
requirements
 As a quality control, some flasks of this bank (about two or three) should be used to confirm that the cell bank
concentration and viability are satisfactory and free of contaminants

33. 7. Culture quality control and laboratory safety
 The culture system should be totally free of compounds that can cause toxic or inhibitory effects
 Contaminants in medium –
Sr. No. Components Effect
1 High concentrations of a culture medium
component in culture
Growth inhibitory
2 Contaminants in medium components and in the water
employed for culture media formulation
Toxic for medium
3 Flasks that have not been well rinsed Toxicity for cell culture
4 Light (particularly short wavelength) can interact with
certain culture medium components, such as riboflavin,
tyrosine, and tryptophan
Generate toxic products

34.  Obtaining cell lines from known and safe sources is important to guarantee the use of well-characterized and authentic
cells with respect to DNA profile, species of origin, and contaminants
 The main contaminant types are –
Sr.
No.
Type of contamination Observation
1 Bacterial and fungal contamination Rapid increase in medium turbidity or by a rapid pH change
2 Mycoplasma contamination Most difficult to detect and can cause a reduction in cell growth
rate, morphological alterations, chromosomal aberrations, and
changes in metabolism of amino acids and nucleic acids
3 Virus contamination Causes changes in cell growth rate
Fungi Mycoplasmas VirusesBacteria
 Fetal bovine serum is usually the main source of viral contamination

35.  Some cell lines are immortalized employing virus, but these cases are not considered as contaminations
 When contamination occurs, it is recommended to discard the culture and continue working with contaminant-free
stocks
 Antibiotics could be used to try to eradicate the contamination but viral contaminations cannot be treated and they
are intracellular parasites
 If virus-free stocks do not exist, a risk evaluation should be performed before continuing to work with the infected
cell line
 Major risks are related to potential injuries resulting from liquid nitrogen manipulation or glassware accidents

36. 8. Characteristics of the main cell lines employed industrially
 The cell lines most commonly employed for production of biopharmaceuticals include CHO-K1, BHK-21, and Vero
cells, besides various antibody-secreting hybridomas
 The CHO-K1 (Chinese hamster ovary) cell line was established by Puck et al. (1958), and was isolated from a
Chinese adult hamster
 The CHO-K1 cell line consists of epithelial cells that can be adapted for suspension growth
 The BHK-21 (baby hamster kidney) cell line consists of adherent fibroblasts, that can also be adapted to suspension
culture, and was isolated from five 1-day-old hamsters (McPherson and Stoker, 1962)
 These cells are commonly used for virus propagation (polio, rabies, and foot-and mouth disease) for production of
veterinary vaccines
 The NS0 cell line, derived from mouse myeloma cells, is one of the most
 The popular for heterologous proteins expression on a large scale due to its capacity for incorporating exogenous
DNA and stably producing recombinant proteins

37.  PER.C61 cells have been used extensively for the production of gene therapy vectors
 HEK-293 cells, derived from human embryo kidney, were transformed with human type 5 adenovirus
 The MRC-5 cell line, derived in 1966 from normal human lung tissue (Jacobs et al., 1970), is adherent and shows a
fibroblast morphology
 These cells are well known owing to their susceptibility to several virus types, being employed in assays related to
viral transfection, in cytotoxicity evaluation, and in vaccine production