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