Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy

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where  refers to speciﬁc cell growth rate, X is cell concentration and t is

the culture time.

The lag phase occurs after cell inoculation. In this phase, there is no cell

division or division takes place at low speciﬁc rates. It is an adaptation

period in which 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 (Freshney, 2005). The duration of the lag phase is dependent

on at least two factors: the point in the growth phase from which cells

were taken in the previous culture and the inoculum concentration. 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 and hence increase the dura-

tion of the lag phase, which is not desirable.

Exponential or log phase is a period of active proliferation during which

the cell number increases exponentially. In the log phase, the percentage of

cells in division can reach 90–100%, and cells are in their best physiologi-

cal state, which is ideal for cell function studies. During the log phase,

there is a kinetic proﬁle typical of all cell lines. The cell doubling time (t

)

can be determined through the direct integration of equation 1, since  is

constant and, in this phase, attains a maximum value (

max

), resulting in:

ln 2



max

(2)

Culture time

Log (cell concentration)

CBA

µ 0⫽

µ 0⫽ µ 0⫽

µ ⫽

max

Figure 2.5

Normal animal cell growth curve pattern, in which  is the speciﬁc cell growth

rate. Lag phase (A) represents the culture adaptation period, followed by an

exponential cell growth phase (B) until the attainment of a stationary or plateau

phase(C),inwhichthereisnoincreaseincellnumber.Theculturereachesthe

senescence p hase (D) when the percentage of cells in division becomes lower than

thepercentageofcellsdying.

22 Animal Cell Technology

Factors that inﬂuence the duration of the log phase are inoculum

concentration, cell growth rate, nutrient availability, and accumulation

of inhibitory metabolites. For adherent cells, the end of the log phase

may also occur at conﬂuence, when cells cover all the available

growth surface, at which point contact inhibition restricts further

growth.

During the stationary or plateau phase, cell growth rate is reduced

due to low nutrient concentrations and the accumulation of inhibitory

metabolites. In some cases, proliferation almost completely ceases. Dur-

ing this phase, cell division is equilibrated with cell death, and the

percentage of cells in division is at most 10%. At conﬂuence the growth

of adherent cells is inhibited by cell to cell contact but a certain degree

of mitotic activity may still be observed. Cells occupy a smaller surface

area, exposing less of their own membrane surface to the culture

medium. A relative increase in specialized protein synthesis (as opposed

to structural proteins), as well as a change in cell surface composition

and charge modiﬁcation may occur (Freshney, 2005). The stationary

phase may be prolonged if the culture medium is replenished with fresh

medium. This is not a stable period for most cell lines, and they are

more susceptible to injuries.

The stationary phase is followed by a decline period in which cell death

is not compensated by cells in proliferation. Cell death can occur by two

distinct mechanisms, named necrosis and apoptosis. Necrosis occurs as a

result of an irreversible injury and normal homeostasis is lost. In vivo, this

form of death generally affects the neighboring cells and may result in

inﬂammation. Autodestruction occurs by activation of hydrolases when

there is a lack of nutrients and oxygen, followed by progressive disorgani-

zation and complete disintegration of the cell.

Apoptosis, on the other hand, occurs through the activation of a

biochemical program involving a cascade of cell components, which is

internally controlled, requiring energy and not involving inﬂammation in

vivo. The most frequently observed biochemical events during apoptosis

comprise caspase activation, mitochondrial membrane permeation, leakage

of diverse molecules from the mitochondria, nuclease activation, cytoske-

leton destabilization, externalization of phosphatidylserine to the outer

membrane, and protein interconversion. This topic is discussed more

extensively in Chapter 7.

The determination of the cell growth proﬁle is important to evaluate the

speciﬁc characteristics of a cell line culture. Cell behavior and biochemis-

try are signiﬁcantly altered in each growth phase. Hence, knowing the

growth curve of each cell line is important for establishing the most

adequate inoculum concentration, prediction of the length of an experi-

ment, and the most appropriate time intervals for sampling.

Cell concentration in suspension can be determined through an optical

microscope employing a hemocytometer for manual cell counting, or in a

semi-automatic way using an electronic particle counter (such as a Coulter

counter), as described in detail by Freshney (2005). Through dye exclusion

(such as trypan blue), it is possible to determine viable cell concentration,

that is the number of cells in a known sample volume capable of

proliferating in favorable culture conditions.

Animal cells: basic concepts 23

2.5 Inﬂuence of environmental conditions on animal cell culture

Cell culture systems must provide the physiological conditions for cell

survival and proliferation. In vitro, animal cell growth is dependent on

several factors, such as pH, temperature, osmolality, gas concentration

(oxygen and CO

), available surface substrata, and state of the cells at

inoculation (Freshney, 2005). Other factors that impact the culture are

medium composition, which can differ extensively between cell lines and

is discussed in detail in Chapter 5, as well as susceptibility to hydrody-

namic stress, as discussed in Chapter 7.

2.5.1 pH

pH control is fundamental for in vitro cell culture. Most mammalian cell

lines proliferate at pH 7.4. Although the optimal pH value for cell growth

does not vary too much for different cell lines, some normal ﬁbroblasts

proliferate well at a pH range between 7.4 and 7.7, and transformed cells

have an optimal growth at a pH varying from 7.0 to 7.4. Insect cells show

better proliferation at lower pH values, from 6.2 to 6.5.

It is possible to monitor pH variation by adding a pH indicator. The

compound most used is phenol red, which is rose-colored at pH 7.8, red at

pH 7.4, orange at pH 7.0, and yellow at pH 6.5. Nevertheless, it is

convenient to point out that most commercially available phenol red

contains impurities that could inﬂuence cell behavior. Also, this com-

pound 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 CO

and lactic

acid derived from glucose metabolism. Most culture media employed for

animal cells are buffered with CO

originating from the gaseous phase, in

equilibrium with sodium bicarbonate (NaHCO

) added to the culture

medium, as described by the following equilibrium reaction:

þ H

O $ H

$ H

þ HCO



(3)

NaHCO

$ Na

þ HCO



(4)

An increase in CO

tends to increase H

and HCO

–

in reaction (3),

consequently increasing medium acidity. In compensation, the increase in

HCO

–

causes NaHCO

formation through reaction (4), until an equili-

brium is reached at pH 7.4. In summary, a medium pH decrease due to a

increase in the gas phase is neutralized by the action of sodium

bicarbonate, with the pH stabilizing at 7.4. Traditionally, culture media

are buffered with sodium bicarbonate at a ﬁnal concentration of 24 mM.

When cells are growing at low densities or are in a lag phase, they do

not produce CO

in sufﬁcient quantities to maintain the pH at an optimal

value. Decreasing the CO

content in the gaseous phase results in an

increase in the culture equilibrium pH. Therefore, the control of CO

concentration allows appropriate maintenance of culture pH (Kilburn,

1991).

This type of buffering is of low cost, non-toxic, and also provides other

chemical beneﬁts for the cells. Each basal medium has its own bicarbonate

24 Animal Cell Technology

concentration and CO

tension recommended to attain the correct pH

and osmolality values. Table 2.2 indicates the recommended NaHCO

and CO

concentrations for a set of commonly employed basal media.

The gas phase in contact with cell culture in an incubator is usually

adjusted to 5–10% of CO

in volume (90–95% air).

In some situations, the utilization of a system with a higher buffering

capacity is needed. In this case, organic buffers can be employed, and in

this category, the most widely used is Hepes (N-2-hydroxyethylpipera-

zine-N9-2-ethanesulfonic acid). Buffering with Hepes is most effective at

the 7.2–7.6 pH range, and this compound is more resistant to rapid

alterations in culture conditions. When using Hepes, a controlled gaseous

atmosphere for mammalian cell growth is not required. Some media are

efﬁciently buffered by Hepes and bicarbonate mixtures. Nevertheless,

Hepes is relatively expensive, and is toxic for cells in concentrations above

100 mM.

For insect cells, culture medium is buffered with sodium phosphate, and

the use of CO

or pH indicators is not required.

2.5.2 Osmolality

Most cell lines present a wide tolerance range to osmotic pressure. Given

the osmolality of human plasma (290 mOsm/kg), it may be reasonable to

assume that this is an optimal value for human cells cultivated in vitro,

although it can be different for cells from other species. As a general rule,

the optimal osmolality range for mammalian cells in culture is from 260 to

320 mOsm/kg, while for insect cells higher values are optimal, from 340 to

390 mOsm/kg.

In laboratory and industrial practice it is important to verify the

osmolality of all culture media after alterations in their basic formulation

Table 2.2 Recommended values of NaHCO

in the liquid phase and CO

concentration in the gas phase to be employed with traditional basal media

Basal medium NaHCO

concentration

in medium (mM)

percentage in gaseous

phase

Mammalian cells culture media

Eagle MEM* 4 Atmospheric air

IMDM 36 5

TC199 26 5

DMEM/Ham’s F12 29 5

RPMI 1640 24 5

Ham’s F12 14 5

DMEM 44 10

Insect cells culture media

Grace* 4 Atmospheric air

IPL-41* 4 Atmospheric air

TC100* 4 Atmospheric air

Schneider* 4 Atmospheric air

From Davis, 2002.

*With Hanks’s balanced salts solution.

Animal cells: basic concepts 25

due to the addition of salt solutions, supplements, pharmaceuticals, and

hormones, or large quantities of buffering agents. Also, metabolic trans-

formations that occur during cell culture can cause osmolality changes.

Culture medium osmolality can also increase due to evaporation, since

culture ﬂasks are generally not sealed so as to allow equilibrium be-

tween culture medium and the CO

–air gas mixture. A slightly hypo-

tonic culture medium can be more adequate for open cultures in

multiple well plates or in Petri dishes to compensate for evaporation

during incubation. To avoid large variations in osmolality during

culture, the relative humidity of the culture environment should be

maintained near to saturation.

2.5.3 Temperature

Temperature plays a very important role in cell culture. It has a critical

inﬂuence on cell growth and it affects the solubility of various medium

components, especially of gases such as CO

and O

which have low

solubilities. Most mammalian cells have optimal growth rates within the

range of 35–378C. Insect cells grow at optimal temperatures of 26–288C,

while cold-blooded vertebrate cells normally grow well at lower tempera-

tures.

2.5.4 Oxygen supply

The most important components of the gaseous phase are oxygen and

carbon dioxide. Monitoring and controlling these gases in culture medium

are essential for in vitro animal cell culture.

Cultures vary signiﬁcantly with respect to oxygen demand, but in

general, 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 frequently the ﬁrst component to be limiting in high cell

densities due to its low solubility in aqueous medium. Aeration should be

performed gently and can be done by the following methods, either

directly or in an isolated vessel: surface aeration, gas bubbling, diffusion

through membranes, increasing O

partial pressure and atmospheric

pressure.

For surface aeration of static cultures, in a closed system, a void volume

10 times larger than the culture medium volume is necessary for adequate

oxygen supply. In agitated cultures with surface aeration, oxygen transfer

is dependent on agitation rate and impeller geometry.

Gas bubbling is a simple and efﬁcient way to transfer oxygen. Never-

theless, this procedure can cause damage to animal cells due to bubble

formation. Alternatively, oxygen can be efﬁciently transferred to culture

medium through hydrophobic membranes (silicon tubes), but this meth-

od 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 O

partial pressure also increases

oxygen solubility and its diffusion rate, but it should be employed only

26 Animal Cell Technology

at high cell density, otherwise oxygen toxicity effects could occur.

Although oxygen is essential for cell growth, it can be toxic in high

concentrations.

Nonstatic cultures of animal cells are common with agitation and

aeration performed as associated operations. These processes, allied to the

fact that animal cells do not possess a cell wall, can result in cell damage if

they are intense, provoking effects that alter cell metabolism, cell cycle,

DNA synthesis, and protein expression, and induce cell death by apoptosis

or necrosis.

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. Many additives, such as serum and the surfactant Pluronic

F68,

have been employed since the 1950s to protect cells from ﬂuid-mechanical

forces.

Serum is known as a good cell protector agent (Cartwright, 1994), and

this effect is normally attributed to the increase in medium viscosity.

However, since there is a strong tendency to eliminate serum from current

technological applications of animal cell cultures, the use of methylcellu-

lose and carboxymethylcellulose as serum substitutes has shown very

promising results. For more complex cases, in which ﬂuid-mechanical

protection is required, as in bioreactors with intense agitation and bub-

bling aeration, the use of Pluronic

F68 at 0.1% (v/v) concentration can

result in an efﬁcient cell-protecting effect.

2.5.5 Composition and nature of the substratum for cell adhesion

Adherent animal cells (anchorage-dependent) need a surface for adhesion,

spreading, and proliferation. Glass and plastic are the most common

materials employed as a solid substratum.

For several decades, glass was the only substratum used for animal cell

culture. As a consequence, the common term ‘‘in vitro’’ means ‘‘in glass.’’

Animal cells present good adhesion on glass surfaces, especially borosili-

cate surfaces, that have a high silica content. Glass is still used as a solid

substratum on a small scale, but very rarely in cultures aiming at high cell

quantities.

The use of plastic materials for routine cell culture on the laboratory

scale was introduced at the end of the 1960s, and some characteristics of

glass surface, such as hydrophobicity and negative charge, were main-

tained in these materials. Polystyrene is the most widely used plastic

material for animal cell adhesion at present, because of its surface charac-

teristics, its low cost, and also its transparency. For more demanding cell

lines, the surface has to be submitted to a treatment that involves coating

with proteins such as poly-lysine, poly-ornithine, or extracellular matrix-

derived proteins such as ﬁbronectin, laminin, and collagens.

The use of microcarriers as substrates is recommended for adherent cell

culture on larger scales. Microcarriers are generally spherical particles that

provide a large effective area for cell adhesion, allowing adherent cell

culture in homogeneous agitated systems. Microcarriers can be produced

from a variety of materials such as glass, dextran, agarose, collagen, or

modiﬁed polymers, and are easily recovered. The same requirements to

Animal cells: basic concepts 27

obtain adequate cell adhesion and growth in culture ﬂasks are applicable.

Cell growth characteristics and animal cell culture applications on micro-

carriers are further discussed in other chapters of this book.

2.6 Cryopreservation and storage of cell lines

Cells can be unstable when in culture for long periods, exhibiting altera-

tions in their morphology, function, growth pattern, and karyotype. Ani-

mal cells show in vitro changes related to age and some cultures can suffer

spontaneous transformation, showing altered growth or changes in func-

tional characteristics. When adequately frozen, cells can be preserved for

long periods without alterations in viability or in other characteristics.

Thus, a cell bank, consisting of frozen and stored cell lines, makes it

possible to maintain the cultures as a renewable source. This procedure

enables repeated cultures to be performed with cells having equivalent

characteristics, with consistent passage numbers. Cells stock also mini-

mizes 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 CO

incubators.

Culture conditions inﬂuence the survival of cells submitted to cryopres-

ervation. To be frozen, cells should be in an active growth phase, with a

viability greater than 90% and free of contaminants. The optimal cryopres-

ervation conditions are different for each cell line. When a cell is exposed

to low temperatures, ice crystals are generally formed and can disrupt the

cell membrane, causing death. Therefore, cells should be treated with a

cryoprotector to support the freezing and thawing processes.

The optimal cell concentration in a suspension of cryoprotector medium

depends on the cell type and is determined empirically, but is generally

within the range of 1 3 10

to 1 3 10

cells/ml. When added to medium at

concentrations between 5 and 10% (v/v), cryoprotectors such as glycerol

or dimethylsulfoxide (DMSO) affect membrane permeability, allowing

water release from the cell interior during cooling. Cryoprotectors de-

crease the freezing point, and ice crystals start to be formed at –58C

Water

efﬂux plays a key role in the freezing process. With cell dehydration,

occurring at between –58C and –158C, ice crystals are formed around the

cells and not inside them. The cooling velocity is also critical and should

be low (about 18C/min). Freezing conditions should minimize crystal

formation in the cell interior, preventing lysis. For cells cultivated in the

presence of serum, the cryopreservation medium should also contain fetal

bovine serum to protect cells from the stress associated with the freezing

and thawing processes. Generally, serum is added at concentrations above

20%, and can attain 90% of volume of the cryopreservation medium.

Frequently cells are stored immersed in liquid nitrogen at –1968C.

However, storage in vapor phase liquid nitrogen (at –1408C to –1808C) is

recommended, avoiding possible contaminations or cell death in case of

cryotube rupture.

During cell thawing, the heating rate should be high. As a general

practice, ﬂasks containing frozen cells are immersed in water at 378C, and

thawing occurs in about 90 seconds. Some cells are particularly susceptible

28 Animal Cell Technology

to osmotic shock during thawing and culture medium transfer processes.

Routine protocols normally describe a dilution stage (1:10 to 1:20 v/v), in

which fresh culture medium should be gradually added to the thawed cell

suspension. Alternatively, cells can be suspended initially in a solvent that

does not permeate the cells, equimolar to the cryoprotector, which would

allow its diffusion from cell membrane before transferring the cells to an

isotonic medium.

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. Then, a ﬂask of the bank should

be thawed and the cells cultured to obtain a working bank. The size of this

bank will depend on the future demand. At this stage it is important to

conﬁrm that the master and the work banks are genetically identical.

Cell bank management insures the maintenance of the cell line original

characteristics, consistency, use of cells with the same passage number,

and also the availability of the cells for culture when required.

2.7 Culture quality control and laboratory safety

Cell culture practice requires rigorous control with respect to the quality

of material and reagents, the origin and integrity of the cell lines, and the

absence of microbial contamination.

The culture system should be totally free of compounds that can cause

toxic or inhibitory effects. High concentrations of a culture medium

component in culture may be growth inhibitory, even if only by increasing

the osmolality. Contaminants in medium components and in the water

employed for culture media formulation can also be toxic. Flasks that have

not been well rinsed can expose cells to signiﬁcant quantities of detergents

or other components that are prejudicial for cell culture. Light (particu-

larly short wavelength) can interact with certain culture medium compo-

nents, such as riboﬂavin, tyrosine, and tryptophan, and generate toxic

products.

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.

Microbial contamination is the most frequent problem in cell culture.

The main contaminant types are bacteria, fungi, mycoplasmas, and viruses.

Bacterial and fungal contamination is detected by a rapid increase in

medium turbidity or by a rapid pH change. After contamination, animal

cells generally survive for a short period. Mycoplasma contamination is

the most difﬁcult to detect and can cause a reduction in cell growth rate,

morphological alterations, chromosomal aberrations, and changes in meta-

bolism of amino acids and nucleic acids. Virus contamination causes

changes in cell growth rate, and fetal bovine serum is usually the main

source of this kind of contaminant. Some cell lines are immortalized

employing virus, but these cases are not considered as contaminations.

Animal cells: basic concepts 29

Another problem that is underestimated in animal cell culture is the

routine use of antibiotics. Continuous utilization of these compounds is

not advised because it favors the development of resistant microbial strains

that become difﬁcult to eradicate, requiring the need for potent antibiotics

that could be more toxic to the animal cells. Furthermore, the use of these

compounds tends to mask contamination at reduced levels.

When contamination occurs, it is recommended to discard the culture

and continue working with contaminant-free stocks. If this is not possible,

antibiotics could be used to try to eradicate the contamination. Neverthe-

less, viral contaminations cannot be treated since viruses do not respond to

antibiotics. Removing them by centrifugation or other separation techni-

ques is not possible, since they are intracellular parasites. If virus-free

stocks do not exist, a risk evaluation should be performed before continu-

ing to work with the infected cell line.

A cell culture laboratory, in which activities are restricted to manipula-

tion of established or pathogen-free derived cell lines, is a relatively safe

workplace. Major risks are related to potential injuries resulting from

liquid nitrogen manipulation or glassware accidents.

When developing activities involving cell lines carrying pathogens or

primary cultures derived from infected animals, there is a potential risk of

operator infection. Viruses present the highest contamination risk, but

many bacteria, fungi, mycoplasmas and parasites can also be harmful to

the operator.

Continuous cell lines not derived from humans or primates and well-

characterized diploid human cells lines with a ﬁnite lifespan, for example

MRC-5 cells, are considered low risk cell lines. Poorly characterized

mammalian cell lines are classiﬁed as medium risk cells. Among high risk

cell lines, are human and primate tissue cells, cell lines carrying endogen-

ous pathogens, and cell lines manipulated after experimental infections.

2.8 Characteristics of the main cell lines employed industrially

The cell lines most commonly employed for production of biopharmaceu-

ticals include CHO-K1, BHK-21, and Vero cells, besides various anti-

body-secreting hybridomas. Other cell lines, such as NS0, HEK-293 and

PER.C6

, have also been employed for recombinant protein production.

The CHO-K1 (Chinese hamster ovary) cell line was established by

Puck et al. (1958), and was isolated from a Chinese adult hamster. It was

one of the ﬁrst mammalian cell lines developed with success for utilization

in recombinant protein production. The CHO-K1 cell line consists of

epithelial cells that can be adapted for suspension growth. These have

become the major cell type employed for recombinant protein production.

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.

Vero cells, isolated from adult African green monkey kidney, consist

of adherent ﬁbroblasts that show continuous growth (Yasumara and

30 Animal Cell Technology

Kawakita, 1963). This cell line is certiﬁed by the WHO (World Health

Organization) and is usually employed for virus propagation (polio,

rabies) for human vaccine production. Another important application of

these cells is in cytotoxicity studies of biomaterials projected for repairing

or reconstituting injured human tissues.

Hybridomas, described initially by Ko

hler and Milstein in 1975, are

immortal cells generated by the fusion of myeloma cells (immortal tumor

lymphocytes that lose their capacity for producing antibodies) with

normal B lymphocytes commonly obtained from rodent spleen and that

have a limited life cycle before the fusion process. Among the major

fusion-promoting agents employed for hybridoma production are poly-

ethylene glycol (PEG) and Sendai virus. Procedures for obtaining hybri-

domas producing monoclonal antibodies (mAbs) are discussed in detail in

Chapter 17.

The NS0 cell line, derived from mouse myeloma cells, is one of the most

popular for heterologous proteins expression on a large scale due to its

capacity for incorporating exogenous DNA and stably producing recom-

binant proteins. This cell line grows in suspension in a highly dispersed

form, without clump formation. NS0 cells show robust growth with the

production of high levels of proteins in different types of culture media.

Nevertheless, when used for recombinant antibody expression, these cells

may incorporate an antigenic glycan residue into the immunoglobulins

that in some cases can result in adverse immunogenicity from interaction

with about 1% of circulating antibodies in humans.

PER.C6

cells have been used extensively for the production of gene

therapy vectors. This cell line was derived from the immortalization of

human embryonic retina cells through the use of adenovirus E1 gene

(Fallaux et al., 1998). This cell line has been well characterized since its

establishment, and no retroviruses or adventitious viruses have been

detected in it. This cell line is easily adapted to different growth conditions

and stably produces high levels of recombinant proteins.

HEK-293 cells, derived from human embryo kidney, were transformed

with human type 5 adenovirus (Graham et al., 1977). These cells exhibit

epithelial morphology and can be adapted to suspension growth in serum-

free media. In addition, this cell line is easily transfected and has been

explored for viral vector production for gene therapy and for obtaining

human recombinant proteins with normal glycosylation proﬁles.

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.

2.9 Culture of insect cells

Insect cell culture was started in 1915 but no cell line capable of replicating

indeﬁnitely was established until 1962. In that year Grace showed the

ability of the insect cell line Antheraea eucalypti to replicate indeﬁnitely in

Animal cells: basic concepts 31