CelIculturennd

biotechnology
Objectiaes
After reading this chapter you should be able to:
I describe the ways in which eukaryotic cells behave when gtown in culture;
I outline some of the strategies used when cell culturós are scaled-up for the
industrial production of biologicals;
I appreciate the principle behind the commercial production of biologicals
using cell culture.

3.L Introduction
Cell culture involves the growth and maintenance of individual cell types in il Plant tissue culture is normally used as a
aitro. Cells in whole plants or animals exist in an organized tissue matrix blanket term to cover the cultiíation in
(Chapter 8) and are influenced by chemical signals such as hormones and controlled environmental condiüons ín
growth factors (Chapter 10) which allow for their controlled growth and aitro of aLlplant parts whether single cell,
a group of cells or an org¿rn.Animal
differentiation. In order to establish a cell culture these cells must be isolated tissue culture involves tlre growth of
from the tissue matrix and incubated in culture media containing a suitable cells in a tissue matrix. In tñis chapteq
mixture of nutrients and growth factors. cell culture is used to mean the grbw&of
Cell culture is now a well established technique and is widely used for isolated cells.
several purposes. For example, the responseof selectedanimal cells to the
addition of chemical agents has led to a better understanding of hormone
acüon or the effects of toxic substances.Of particular importance in the last
few years has been the use of cell cultures for the productio¡ of coÍunercially
valuable products, termed biologicals. This has been one of the major
advances of modern biotechrrology.
Biologicals are single molecules or complex mixtures of compounds
derived from living systems which have application in human and animal
healthcare. In particular, the ability to produce biologicals, such as vaccines,
routinely and cheaply from cell cultures has led to a great improvement in
Explant
public health during the last 40 years.
Coverslip
The number of these biologicals is expanding rapidly and numerous
biotechnology companies have grown ready to exploit the available
technology in manufacturing processesdesigned for large-scale eukaryotic
cell culture. However, growing cells in culture is not easy and a great deal of
painstaking experimentation went into establishing the conditions necessary
for the continuous culture of cell lines.
Fig.3.f An illustration
of Harrison's
hanging
droptechnique(1907). Thiswas oneof the first
methodsreportedfor growingeukaryoticcellsrn
3.2 Thebeginningsof animaland plant cell culture vitro.

Several individuals were associatedwith the development of cell culture

biological : a commerciallyiseful proiluctfrom ReferenceMorgan, S.I.and Darling, D.C.

cells.lt may bea singlecompoundor a complex (1993)Animal CeIICulture,Biqs,Oxford, UK. Introduction
87
mixture. This text describes practical details as well as
the fundamentals associatedwith cell culture.
Table3.7 Milestonesin thecultureof animaland plant cellsfor productionof biologicals

Animal cells Plant cells

1883 Cell theory proposed

Chick embryo cells maintained in vitro in saline 1885

Frog nerve cells cultivated using'hanging drop' 1900-1910 Totipotency postulated, attempts at plant cell culture
Aseptic techniques introduced
193U7940 Indoleaceticacid (plant hormone) isolated

Continuous cell line from human cervical carcinoma cells 1950-1960 Study of rubber biosynthesis in guayule callus pioneered
established (HeLa cells) Auxin/cytokinin interaction found necessaryfor
First widely used chemically defined medium (Eagle's) organogenesis
Early efforts to grow cultures in millilitre suspensionsystems

Human fibroblasts shown to have a finite life-span in culture L96Vr970 lnorganic growth medium investigated
HAT medium used for cell selection
Serum-free medium introduced
Human and mice cells fused by use of a virus

Monoclonal antibody-secreting hybridomas produced L970-1980 Continuous culture of cell suspensions

Organ cultures developed that produced many different com-
pounds
Semibatchculture of tobaccocells scaled-up to 200@ dm"
fermenters
Digitoxin biotransformed to digodn by tissue cultures of
Digitalis sp. (foxglove)
Importance established of selecting high-yielding cells from
non-producers of biologicals

Recombinant DNA technology allows large-scale production 1987-1995 Development of immobilized cell systems -
of biologicals fron\ genetically engineered cells Study of factors iñfluencing expression gf secondary
metabolites in cultures cells

t987 Hair-like root system transferred by Agrobocteriumrhizogmes

used as cell cultures in bioreactors for producing secon&ry
metabolites
:
1990 Thxol, an anti-cancercompound, was
isolated from cell cultures of Taxus
Nine animal cell culture products are licensed for human ther- 1987-1993
apeutic use (seeTiable3.8).

Box3.1 The originalimpetusfor the appl¡cat¡on of cellculturetechniquesfrom a laboratory

toalargeindustria|Sca|earosefromtheabi|itytogrowV¡rUSeSinanima|ce|ls
Vaccineproduction 1949it was shownthat the poliomyelitis viruscouldbe grown in cellculture.This
findingestablished the basisfor the developmentof a large-scaletechnologyfor
vaccineproduction. The.poliovaccinewas producedfor masshumanvaccinat¡on in
the 1950s.Sincethat time a rangeof otherbiologicals synthesizedfrom an¡mal
cellshavefoundcommerc¡al application
and havebeenresponsible for muchof
the scientificand financialinterestin modernanimalcellbiotechnology.

microbial contamination although it is usually necessaryto add serum to

provide growth factors, some as yet probably unknown.
Media formulations used today include a complex mixture of
carbohydrates,amino acids, salts,vitamins, hormones and growth factors.An
example of an extensively used medium is shown in Table3.2.
The salt concentration is isotonic to prevent osmotic imbalances.
Bicarbonateis often included to act as a buffer system in conjunction with the
carbon dioxide-enriched environment (5-10% CO2/95%air) in which the cells
arc cultured. This allows cultures to be mafrrtained around the optimal pH for
growth of about 7.4. Phenol red is normally included in the medium as a pH

isotonic medium: a culture medium,theosmotic

pressureof which is thesme osmoticpotentialas Animalcell.culture89
intracellularfluid.
However, the use of antibiotics for routine subculture of stock cultures should
be discouraged because low level contamination may be masked and may
cause problems at a later date. In addition, a number of these antibiotics are to
some extent cytotoxic. Careful aseptic handling techniques can ensure a low
risk of contamination.

The characteristics of animal cells in culture

A primary cell culture is established by inoculating cells taken directly from

animal or human tissue into growth medium. The tissue is gently fragmented
into small pieces and these are placed in a sterile medium in a petri dish.
Treatment of the excised fragments with a proteolytic enzyme disaggregates
the tissue into individual cells.
Cell types may be recognízedby their characteristic shapes when examined
using a microscope (Fig. 3.5). Thus,fibroblasts, which are spherical when first
treated with trypsin, elongate to a spindle shape on attachment to a surface.
Epithelial cells, which also have a requirement for surface attachment, have a
characteristic cobblestone appearance.

F i g . 3 . 5 M o r p h o l o g yo f a n i m a l c e l l t y p e sc o m m o n l y g r o w n i n c u l t u r e .S c a n n i n ge l e c t r o nm i c r o g r a p h so f
b o v i n e( a ) f i b r o b l a s t(sx 8 2 5 ) a n d ( b ) e p i t h e l i a l c e l (l sx 7 5 0 ) b o t hg r o w i n go n c o l l a g e nf i b r e s .C o u r t e s y
D r P M . K u m a r ,D e p a r t m e n to f B i o l o g i c aSl c i e n c e s ,t h e M a n c h e s t e rM e t r o p o l i t a nU n i v e r s i t yU , K.
( c )N e u r o np h o t o g r a p h e db y c o n f o c a lm i c r o s c o p y C . o u r t e s yP r o f e s s o rA . B o y d e ,D e p a r t m e n to f
A n a t o m ya n d D e v e l o p m e n tB i o l o g y ,U n i v e r s i t yC o l l e g e ,L o n d o n ,U K . ( d ) P h a s ec o n t r a s t
p h o t o m i c r o g r a po h f h y b r r d o m ac e l l s ( m a d eb y f u s i o no f a m o u s e m y e l o m ac e l l w i t h a m o u s e s p l e e n
c e l l )( x 1 7 6 4 1C . o u r t e s yM s C . R .C o l e ,D e p a r t m e n to f B i o l o g i c aSl c i e n c e s ,t h e M a n c h e s t e r
M e t r o p o l i t a nU n i v e r s i t y U
, K.

Most cells derived from animal tissues require a substratum for growth and
are anchorage-dependent. However, cells associated with biological fluids,
for example blood cells, are non-anchorage-dependent and may be grown in
suspension. Lymphocytes are non-anchorage-dependent cells commonly
grown in culture.
Further cultures may be established by subculturing a primary culture. In
the case of suspension-grown cells, such as lymphocytes, this involves

fibroblast: a celltypefoundin connectiuetissue Reference Butler, M. and Dawson, M. (eds)

-t
in associntionwith collagen (1992) CelI Culture Labfax, Bios, Oxford, UK. A ni malcel lcul ture91
epithelial cell: a celltypederiued
from epithelia, This is a rapid reference source of essential
whichis a layercoaeringan internalor external data for culturing cells
surface.
Table3.4 Animal cell linescommonlyusedin culture

Cell line Origin Characteristics The exponentialgrowth of cells can

be describedby the equation:N =
BHK 9yrur, baby hamster kidney Fibroblasts,anchorage-dependentbut can N0.2"or logN = logNo+ x.log2,where
be induced into suspension N = final cell number,No= initialcell
CHO Chinese hamster ovary Epithelial-like, suspensioncells number,x = ñufrb€r of generations
HeLa Human cervical carcinoma Epithelial-like, suspensioncells of exponentialgrowth. Cpnsidera
IMR-90 Human embryonic lung Diploid fibroblasts, limited life-span t i s s u ee x p l a n to f 1 0 m m ' c o n t a i n i n g
L Mouse connective tissue Fibroblasts,suspensioncells 1 0 ' c e l l s .D e t e r m i n et h e m a x i m u m
McCoy Mouse origin similar to L cells Fibroblasts,anchorage-dependent theoreticalcell yield from the growth
MDCK (Madin Darby) canine kidney Elrithelial-like, anchorage-dependent of such cellsafter 50 generations.
MRC-5 Human embryonic lung Diploid fibroblasts, limited life-span What would be the culturevolume
Mrc-11 Mouse myeloma Lymphoblast-like, suspensioncells necessaryto accommodate such
Namalwa Human lymphoblastoid Lymphoblast-like, suspensioncells growth?
(Burkitt's lymphoma)
3T3 Mouse connective tissue Fibroblasts,anchorage-dependent
wI-38 Human embryonic lung Diploid fibroblasts, limited life-span
WISH Human amnion Epithelial-like, limited life-span
Vero African green monkey Fibroblasts,anchorage-dependent

From Butler, M. (1988)Biotechnology:

A Comprehensiae
Treatise,6b,249-31ó.

Animal cell growth in culture

(a)
Animal cell growth in culture follows a pattern of three distinct phases (Fig.
3.7). The lag phase is a period of zero growth when cells are first inoculated
into growth medium. The length of this phase is dependent upon the type of
cells and their metabolic state at inoculaüon. The exponential growth phase is
a period of continuous cell doubli^g.Animal cells normally exhibit a doubling a
-o)
time of between L5 and 25 hours. Growth continues in batch culture to L-2 x (J

1.06cell cm-3,which is the typical cell density sustainableby presently available -c

media. 3
The stationary phase is a period after growth when there is no changein the o
culture cell density. The phase occurs when the nutrients have been depleted .2

or inhibitory metabolites have accumulated in the culture. Cell death may (b) €
occur in some cell lines by apoptosis, which is characterized by DNA c
fragmentation and the formation of characteristic blebs on the cell surface
o
(Chapter 16). In culture these cells exhibit a rapid decreasein viable cell
c
concentration without an observable stationary phase. Such a'grow or die'
processis characteristicof lymphocyte hybridomas. Further growth of cells
canbe encouraged by subculturing thg cells into fresh mediurn.
The passagenumber of a cell line is recorded as the number of subcultures
from the cells'original isolation. This is useful for monitoring any changes
that might occur to the cells through the period of continuous growth. The
passagenumber can also be related to the generation number of the cell line. Time
This depends upon the split ratio, which is the number of new cultures
established at each stage of subculture. The simplest case occurs Where a Fig.3.7 Growth shown by cells growing in (a)
confluent culture is subcultured into two new cultures; that is, a split ratio of 2. batch and (b) continuousculture. In batch culture
a finite amount of nutrient medium is present and
In this case the generation number and passage number are the same. growth of cells ceaseswhen an essential
Otherwise, generation number - passagenumber X split ra|lLo / 2. nutrient is depleted (A, lag phase; B, exponential
phase; C, stationaryphase).In continuous
culture, cells are constantlysuppliedwith
nutrientsbecauseof the inflow of fresh medium.
Modes of cell culture
The simplest mode of culture is a batch culture in which cells are inoculated
and left for several days until growth stops. This is a closed system because
nothing is added or removed from the culture.'A typical growth pattern
would be the inoculation of cells at 10scm-t and incubátion for-3 days tb ailow
the cells to accumulate to a final cell density of 1.06'cm-3.
Stirring ensuresa'
relatively homogeneous mixture of cells and media components. However,

doubling timc: the time (h) talccnfor a cell passagenumber:thenumberof subcultures(or

Wulation to doublein number. passages) performedafter theoriginal isolationof Animalcellculture 93
apoptosis: cellileathcausedbytheactiaationof thecellsfrom aprimary source.
intracellular degrailntiaeenzryes. This is akin to
'suicide
a mechanism' for cells.(SeeChapter16).
 1x1os

1X108

1x107
f)
I
tr
C)
a
q) 1x106
O

1x105

1x104 bó
Time (day)

Fig. 3.9 The typical final cell densities that can be expected from a batch culture, perfusionculture and
n o r m a la n i m a lt i s s u e s .

3.4 Plant cellculture

Plant cells are totipotent. This means that, given the appropriate nutritional
factors, plant growth regulators and physical environment, it would be
expected that any plant cell should be able to regeneratethe phenotype of the
complete and fully developed organism from which it was originally derived.
(This is not the casewith animal cells.) This implies that it should be possible
to induce plant cells to produce any substancecharacteristic of the parent
plant. Suspensionsof single cells are generally the type of culture chosenfor
production of such compounds.

The culture of plant cells

It is now possible to initiate a cell culture from almost any plant species,
although for reasonsnot futly understood this is easierwith some speciesthan
others. For example, cultures of potato and tobacco cells can be started
relatively easily,whereas cerealsand grassesare, in general,rather difficult. Fig.3.10 (a)Callusof sugarbeet,two months
(x3.5).(b)Sugarbeetcellsfrom
afterinitiation
Cell suspension cultures are obtained from a friable callus (FiS.3.10)which suspension culture,7 daysold (x 190).Courtesy
can readily disperse to give single cells and/ or cell aggregateswhen agitated Dr J. McEntyre,Department of Biological
Sciences. theManchester Metropolitan
in liquid medium. University, UK.
The callus, which arises from the disorganrzed proliferation of cells, can be
derived from meristems or tissue explants. Callus cultures are usually grown
on a solid medium. After a few weeks, actively growing cells can be
transferred into liquid medium for continued growth in suspension culture.
Theseactively growing cell suspensionscan be propagated by subculturing
aliquots to fresh medium at regular intervals, a technique known as batch
propagation. It is important that aseptictechniquesbe maintained throughout
the isolation and culture of plant cells (as,indeed, with animal cell culture) to
prevent
^ contamination with microorganisms.
Typical biomass yields in culture oÍ betr"een 5 and 20 g dry weight per dm3
have been recorded over a growing period of 8 to 10 days. The growth pattern
of plant cells is similar to those of animal cells (Fig. 3.7).

ReferenceStafford, A. ur,a Wut G. (eds) callus:an aggregationof proliferatingplant cells

(1991)Plant celland tissueculture,"n,
Open danelopedin culture. Plantcellculture 95
University Press,Milton Keynes.A readable
text included in the OU PressBiotechnology
seriesand describing the basics of culture
methods.
Screening for high-yielding plant cell culture
Selection and screening for high-yielding clones relies upon the inherent
genetic instability of plant cell populations (that is, on somaclonal aariation).
Withln any plant population, there will be individuals producing much more
of the required natural substance than other cells in that population. Flowever,
variabiliiy and instability within cell populations, whilst highly desirable in
any selection process, gives cause for concern in the maintenance of high-
yielding cultures. These problems have not yetbeen fully resolved.
Higtr product-yielding mutants can also be induced artificially by gy,
MolecularBiology and Biotechnolo
ultraviolet irradiation or chemical mutagens, for example, N-methyl-N-nitro- Chapter 8
N-nitrosoguanidine.
Isolating high-yielding strains from amongst clones of plant cells can be a
difficult task. This is because minute quantities of the desired product are
present in single cells or aggregates of cells in culture. The process is easier if
the desired product is pigmented, since the appropriate cells can be selected
by visual inspection. Yield improvement has been achieved through selection ffi The yield of the red pigment shikonin
from cultures of Lithospermum
and screening together with refinements in the environmental conditions erithrorhizor¿is L5 times greater (weight
during culture. Such improvements have led, in some cases, to plant cell for weight) than that from the whole
suspeñsion cultures producing as much, or more, product (weight for weight) plant. Shikonin, which has antibacterial,
than the whole plant. Table 3.5 summarizes the culture conditions which anti-inflammatory and antifumour
properties in addition to being a
influence the accumulation of secondary metabolites in plant cells. valuable dye, was the first plant tissue
culture product to be commercialized (in
1983inJapan).
ofcultureconditions
Table3.5 Summary theaccumulation
whichinfluence metabolites
ofsecondary in
plantcells
Conditions before culture External culture conditions Internal culture conditions

. disease-freestock plants . light . components of the medium

. use of whole plants which o temPerature o aeration and culture mixing
yield high quantities of . culture vessel agitation . culture medium pH
the desired substance o cell maturity

Interesting reference Brodelius, P. and

Apart from accumulating secondary metabolites, plant cell-scan also be used Pendersen, H. (1993) Increasing
tocatalyse enzymatically the conversion of organic materials to commercially secondary metabolite production in
important biochemicals. The conversion of part of a molecule_ using a plant-cell culture by redirecting
transport. Trendsin Biotechnology,ll,
biological system is called biotransformation. Bacterial uld fungal 30-36.
biotránsformations have been used for a number of years in the large-scale
production of steroids. Plant cell cultures may also be used to achieve such
biotransformations, although they are used to a lesser extent at present. Plant
cell cultures are inherently slow-growing, making the scale-uP Process
expensive. However, there is potential in the development of genetically
engineered bacteria transformed with specific plant genes that would be able
to perform some of the biotransformations.

3,5 Thescale-upof animaland plant cellcultures

Scale-upmeans increasing the capacity of a culture so that a laboratory-based
processmay be developed commercially. The initial stage of scale-up TaI
involve optimizing the production of the cells in culture. The mass growth of
many miirobial systems has been extensively studied and many of the
prinóiples determined in thesesystemscan be used by th9 animal or plant cell
óulturé biotechnologist. However, there are a number of distinct differences
between animal, plant and microbial cells which lead to fundamental
differencesin appróach to the large-scaleculture of thesecells (Thble3.6).

identical cells
clone:a populationo¡gene:tically
derioedfrom an indiaidualcell.
somaclonal oariation: theincreasein genetic
aariabilitywhichtalcesplacewhenhigherplants
areculturedin vitro.
Reference Smith, R.H. (1992) Plant tissue
culture: techniquesand experimenfs,Academic
of cellcultures97
Thescale-up
Press, San Diego. This is a manual of practical
details and exercises in plant cell culture
suitable for undergraduate and graduate
courses.
 Continuous
feed

lmmobilized
ceils Reservoir
Air bubble

Water
Packed jacket
spneres Ai

(c) (d)

Fig' 3.73 Types of bioreactors.(a) Direct-driveslow-speed,stirred tank bioreactor.(b) 2 dm3 airlift Fiq.3.74 Dextran microcarrierscovered witn
f e r m e n t e r .C o u r t e s yL . H . F e r m e n t a t i o nM
, a i d e n h e a dU , K . ( c )A p a c k e db e d b i o r e a c i o r - ( dA) f l u i d i z e d m o n o l a y e r so f m a m m a l i a nf i b r o b l a s t s( x 1 7 b O ) .
led recirculatlng b i o r e a c t o rR
. e d r a w nf r o m M a n t e l l ,S . H . ,M a t t h e w s ,J . A . a n d M a k e e , R . A . ( 1 g g 5 )
P^riryiplesof Plant Biotechnology: An lntroduction to Genetic Enginetering¡n ptaiis. Biackwell Scientific,
O x f o r d .U K .

culture of suspension cells. The airlift fermenter consists of a tall column

which contains an inner baffle or draught tube. Culture mixing is provided by
a stream of air which passes through the inside of the draughl tuLe. Aeratioí
and mixing ne_edcareful control, since concentrations of the[ases, oxygen and
carbon dioxide, may affect biomass and product yield. Such a féñnenter
design_is being used commercially for the proáuction of monoclonal
antibodies (section 3.6) from hybridoma cells grówn in cultures of 1000 dm3
volume.

THE PACKED BED BIOREACTOR consists of a static bed of particles such as

glass beads to which the cells attach and grow. In this system, growth medium
can be continuously pumped through the culture coi.t-t. Íhir system has
been used for the production of foot-ánd-mouth disease vaccine fróm animal
cell cultures.

THE FLUIDIZED BED REACTOR relies on liquid flow to maintain cells in

ReferenceLubiniecki,A.S. (ed.)(1990)tnrge-
ScaleMammalianCellCultureTechnology, Márcel Thescale-up
of cellcultures9g/
Dekker, New York, USA. This multi-authored /
book containsdetailsof proceduresassociated
with the industrial use of mammalian cell
cultures.
Viral vaccines

Viral vaccines produced from animal cell cultures have led to major
improvements in world public health, leading to, for example, the complete
eradication of smallpox and the virtual eradication of poliomyelitis (Fig. 3.16).
Thble 3.7lists viral vaccines in routine use.
The production process for a vaccine involves inoculation of the virus into
the confluent cell culture in which it propagates until a maximum number of
viral particles are obtained. A typical pattern of virus growth in cell culture is
shown in Figure 3.I7. The process can be divided into four distinct stages
related to time bands after addition of the initial viral inoculum.
101 Totalvirus
cr) F i g . 3 . f 6 E l e c t r o nm i c r o g r a p ho f t h e p o l i o v i r u s
F ( x 1 8 60 0 0 ) .C o u r t e s yD r A . C u r r ya n d M s H .
CotterillP , u b l i cH e a l t hL a b o r a t o r yW, ithington
) 1no
H o s p i t a l ,M a n c h e s t e r U
, K.
ci
c
.F
(o Table 3.7 Examplesof airnl aaccinesin common
=
L
lu' use

c Human Veterinary
C)

Polio (Salk)* Foot-and-mouth disease*

.= E x t r a c e lllaur Polio (Sabin) Marek's disease
V US Measles Newcastle disease
Mumps Rinderpest
Rubella Rabies
o 2 4 6 B 10 Yellow fever Canine distemper
(h)
Timeafterinfection Rabies* Swine fever
Influenza Blue tongue
F i g . 3 . 7 2 P h a s e so f v i r a lg r o w t h i n c e l l c u l t u r e .1 , a d s o r p t i o n / p e n e t r a t i o2n,; s y n t h e s ¡ s3; , a s s e m b l y ;4 , Fowl pox
r e l e a s eo f v i r u s e s .
*Produced as inactivated viruses. All othe¡s are
normally produced as live attenuated viruses.

As the maximum extracellular viable virus count occurs in phase 4, it is

important to be able to identify this in order to obtain the optimum viral yield.
The culture medium is concentrated and the virus extracted. The harvested
viruses may then be treated by an inactivating agent, such as formaldehyde, to
render the virus non-pathogenic and suitable for use as a vaccine.

FOOT-AND-MOUTH DISEASE VACCINE is the most extensively used

veterinary vaccine and, in fact, its production level exceeds that of any other
biological. Originally blood extracts from infected animals were used as a
source of vaccine. This was superseded by a process of virus propagation on
bovine tongues obtained from slaughterhouses. The foot-and-mouth disease
virus is now grown in baby hamster kidney (BHK) cells established as a cell
line suitable for continuous growth in fermenters (Fig. 3.18). These cells canbe
grown in suspension or on microcarriers (Fig. 3.1a).

Box3.3 Beforeanyanimalcellproductis approvedfor humantherapeuticuse,strictsafety

standardsmust be observed.ln particula¡it must be shownthat the productis
Thesafetyol fre eo f a n yv i ruscontami nati on andal sodoesnot contai nany D N A .Theabsenceof
productsderiued D N A i s a s a feguard agai nsta tumori geniagent
c contami nati ng the product.
Presenttechniquesallow DNAto be detectedat a level< 1 pg cm-",which is
fro* cellculture severalordersof magnitudelowerthanthe amountof DNA requiredto causea
s i n g l etu m o rigeni event.
c

A ni mal
 The most successful application of cell-cell fusion involves the production
of hybridon ns (Fig. 3.5d) capable of secreting monoclonal antibodies (Fig.
3.19). This process was pioneered by Kóhler and Milstein in the early 1970s
and is also described in Chapter 1,4.It involves the isolation of B lymphocytes
from the spleen of animals (usually mice) which have been injected with an
antigen of choice (Fig. 3.20). These antibody-secreting cells isolated after
immunization are incapable of continuous growth in culture. Immortalization
of these cells can, however, be accomplished by hybridization with a tumour
cell such as a myeloma derived from malignant lymphocytes.
There are three important characteristics of myeloma cells used as fusion
partners in cell hybridiazation:
Antigeninjected

/ \ Mvetoma

M o u s ei m m u n i z e d
ffi;U'Jil,"-
I
S p l e e nc e l l s
removeo
, Fusion
/

+
I

,-1.--, lncubationin HAT

selectsfor HGPRT+
E: (seeBox 3.4)
+ Cellscloned

I Supernatant
assayed
f o rM a b
l-
Positiv e c l o n e s
S Elected
Fig. 3.20 Overview of the production and
/\ t \,^. selectionof a monoclonalantibody-secreting
,Ú
I
h y b r i d o m ac e l l l i n e ( s i m i l a tr o t h a t s h o w n i n F i g .
3 . 5 ( d ) )H
. G P R Th , y p o x a n t h i n eg u a n i n e
é<u 'dt' ProPasationIn ascrtes
rruidor m i c e
phosphoribosyltransferase; HAT,hypoxanthine,
ln vitro \htk a m i n o p t e r i nt,h y m i d i n e ;M a b , m o n o c l o n a l
c e l l propagation \ a n t i b o d y( s e ea l s of i g u r e i n B o x 1 4 . 6 ) .

Box3.4 T h e H G P R T(h ypoxanthi ne

guani nephosphori bosyl transferase)
genei s a
commonlyusedmarkerfor geneticselection.The productof the gene is a
genetic
A selectable transferase enzymewhich functionsin the salvagepathwaymechanismof
marker nucleotidesynthesisandwhich allowsthe nitrogenbases,hypoxanthine and
guanine,to be convertedto theircorresponding nucleotides. Anotherenzyme,
thymidinekinase(TK),is alsoimportantin this salvagemechanismfor the
conversionof thymidineto its nucleotide.The alternative mechanismfor
nucleotidesynthesisis by a de novopathwayfrom simpleprecursors. Normally
cellscan use eitherof thesepathwaysfor nucleotidesynthesis.
Cellswith a defectiveHGPRTenzymecan be selectedfrom cultureby treatment
with 8-azaguanine. Thispurineanalogueis toxicto cellsif incorporated
into DNA
followingmetabolismwith HGPRT.However,HGPRT-cellsare insensitive to the
toxic effects of 8-azaguanine.
HGPRT*cellscan be selectedin cultureby usingthe selectivemediumHAT
(hypoxanthine, aminopterinandthymidine).Aminopterinis a metabolicinhibitor
which blocksthe de novonucleotidepathway,but the presenceof hypoxanthine
and thymidineallowsnucleotidesynthesisto proceedin cellswith a functional
HGPRTenzyme.Thusthe HATmediumis growth inhibitoryto the HGPRTcellsbut
growth promotingto the HGPRT.cells.

ReferenceMilstein, C. (1980)Monoclonal hybridoma: a cellline originatingfrom thefusion

antibodies.ScientificAmerican,243(4),6G7 4. of two parentcellse.g.thefusionof a Blymphocyte
The inventor of monoclonals tells the story. anda myelomacellgiaesriseto antibody-secreting
hybridomas.
 AcD
cells can be grown continuously in culture with a substantial secretion of
interferon into the medium. Such a process is now being conducted in 8000 tI
dm3 batch cultures.
rycHxi
:f

Normal human diploid fibroblasts can be induced to produce B-interferon

by the supplementation of the cultures with a synthetic double-stranded RNA C

(poly I-C) followed by specific antibiotics (Fig. 3.23). The RNA simulates the
effect of a virus and induces the formation of interferon. The stimulation of
.=
cells to produce interferon can be enhanced by the sequential treatment of the
cells with the antibiotics cycloheximide and actinomycin D. The effect of such J

treatment is to increase the life span of the mRNA for interferon, and this
consequently improves the specific productivity.
lrme after removal of inducer (h)

Therapeutic compounds produced from animal cell cultures Fiq.3.23 The superinductionof interferonin
humandioloidfibroblasts.olC
There are numerous compounds produced by animal cell cultures other than (polyinosine:cytosine)
is a syntheticRNA,while
CHx and AcD are the antibioticscycloheximideand
antibodies and the interferons that can be used for human therapy. Table 3.8 actinomycinD, respectively.The antibioticsare
lists some animal cell culture products that have been approved for human addedto inducethe synthesisof interferon.
Redrawnfrom Friedman,R.M. (1981)lnterferons-
therapeutic use following clinical trials. a Primer,Academic Press,NewYork, USA.

licensed
Table3.8 Approaed fromanimalcellcultures
products
Product Full name Application

19ft6 IFN-a Lymphoblastoid interferon Treatment of hairy-cell leukaemia

79f36 OKT3 Anti-CD3 murine monoclonal Prevents tissue rejection in organ
antibody transplants
t987 t-PA Tissue-typeplasminogen Treatment of thrombosis following
activator coronary disease
1989 EPo Erythropoietin Stimulation of red blood cell
development in kidney failure
t989 hGH Human growth hormone Treatment of growth hormone
deficiency (dwarfism)
t989 HBsAg Hepatitis B surface antigen Vaccine to prevent hepatitis
B infection
r991 G-CSF Granulocvte colonv Stimulation of white blood cell
stimulating factoi development following cytotoxic
chemotherapy Fiq.3.24 Photomicrographof CHO cells grown on
t992 FVIII Factor 8 Treatment of haemophilia A surfaceof glassculturebottle.(x145). courtesy
t993 DNase I Deoxyribonuclease Reduces mucus viscosiW in Dr J.A. Wright, Instituteof Cell Biology,University
cystic fibrosis of Manitoba,Canada.

Most of the producing cells are genetically modified by recombinant DNA Exercise
3
techniqueswhich allow the transfer of an isolated geneinto a selectedcell line. ActinomycinD inhibitsthe formation
Chinese hamster ovary (CHO) cells have been used extensively for this of mRNA whereascycloheximide
pulpose and have becomethe first choicefor large-scaleprocesses(Fig. 3.24). inhibitsprote¡nsynthesisfrom
The synthesized products are all complex glycoproteins, which must be mRNA. With this information,
attempt to explainthe mechanismof
synthesized in mammalian cells because of the post-translational interferonproductionfollowingthe
modifications required to ensure full biological activity. These include doubleantibiotictreatment.
proteolytic cleavage,subunit associationor a variety of addition reactions
such as glycosylation, methylation, phosphorylation or acylation. Tissue

Box3.6 Interferonmay be usedtherapeutically in the treatmentof viraldiseasesand some

forms of sancer(Chapter14).Thereare severaltypesof interferonsand their
trnterferon propertiesincludeantiviral, and immunoregulatory
antiproliferative Their
activities.
potentialas therapeuticagentshasnot beenfullyrealizedalthoughthey havebeen
valuablein the treatmentof a limitednumberof typesof cancers.One of the
originalmethodsof obtaininginterferoninvolvedits extractionfrom activated
leucocytesobtainedfrom humanblooddonors.However,this methodwas
( S e ea l s oB o x2 . 11 ) inadequate to supplythe largequantitiesof the compoundrequiredfor treatment.

Animalcellproducts105
 metabolites can be of great commercial value. Examples of secondary
metabolites produced by plants include alkaloids, resins, tannins, sterols,
saponins, volatile oils and cardiac glycosides.
The greatest potential for the use of immobilized plant cells is in the mass
production of such secondary compounds, either by replacing immobilized
enzymes or microorganisms in biotransformations, or in the synthesis of
commercially important substances. However, mass plant cell culture is
initially capital intensive. Therefore, it is not an easy matter to persuade
industry to use novel plant cell culture systems to obtain commercially
important substances as opposed to continued investment in cultivated
plants.
The main limitations of the use of immobilized plant cells arise through
cellular retention of products or a decline in cell viability. Few commercially
valuable natural products from plants are released into the medium; most are
normally retained in a cell vacuole (Chapter 1). Often cell viability is lost
through the use of agents, such as dimethylsulphoxide, to release the
secondary products into the culture medium.
The use of plant cell culture technology as an alternative route to natural
product synthesis does have some distinct advantages. First, it provides
independence from environmental variables such as climate, geography, plant
pests and diseases which may affect product supply. Secondly, a close control
on market supply can be achieved by controlling production, as and when Look aroundyou at home, at work or
required, under defined conditions and thereby giving consistent production. in the street and list some of the
Table 3.9 shows some of the industrial uses of natural plant products. names of productsin common use
Market volumes and costs vary considerably for these products. For instance, that are derivedfrom plants.
the price of the perfumery agent, iasmine, is around eight times that of
quinine (a bittering agent in tonic water and an anti-malarial drug). However,
ffi There are a number of commerciallv
there is a much larger consumer market world-wide for quinine than for
important plant enzymes, includin!
jasmine, at an approximate ratio of jasmine to quinine of 1:L00.There is also a amylases, papain, bromelin, ficin and
large world market for codeine phosphate (an analgesic) and for digitoxin (a pectinases from cereals,papaya,
cardiotonic). Both of these have a consumer demand at about three times more pineapple, fig and citrus fruits,
respectively. All find use in the food
than quinine. industry. For example, papain is used in
As indicated earlier (section 3.4), enzyme systems from cell cultures can be meat tenderization.
used in industrial biotransformations. One of the best-known examples of
such a biotransformation using plant cells on a large scale is that of the
cardiovascular steroid digitoxin obtained from foxglove (Digitalis lanata).The
required biotransformation relies upon a single enzyme-catalysed 12- Definethe terms primarymetabolite
hydroxylation (Fig. 3.26). and secondarymetabolite,as
derivedfromplants.

ofnaturalplantproducts
Table3.9 Examples andtheirindustrialuses
Plant species Plant product Industrial use
Common name Scientific name

Agrochemicals
Pyrethrum Chrysanthemumcinerariaefolium Pyrethrin Insecticide
Cosmetics
jasmine lasminium officinalis Jasmine Perfume
Food and drink
Quinine Cinchonaledgeriana Quinine Bittering agent
Thaumatococcusdanielli Thaumatin Non-nutritive sweetener
Pharmaceuticals
Opium poppy Papaaersomniferum Codeine Analgesic
Yam Dioscoreadeltoidea Diosgenin Antiferülitv
Quinine Cinchonaledgerinna Quinine Antimalarial
Foxglove Digitalis lanata Digoxin Cardiotonic
Thorn apple Daturastramonium Scopolamine Antihypersensitive
Periwinkle Catharanthusroseus Vincristine Antileukaemic

Plantcellproducts107
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