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 culturs are scaled-up for the industrial production of biologicals; I appreciate the principle behind the commercial production of biologicals using cell culture.

nnd CelIculture biotechnology

3.L Introduction
Cell culture involves the growth and maintenance of individual cell types in aitro. Cells in whole plants or animals exist in an organized tissue matrix (Chapter 8) and are influenced by chemical signals such as hormones and growth factors (Chapter 10) which allow for their controlled growth and differentiation. In order to establish a cell culture these cells must be isolated from the tissue matrix and incubated in culture media containing a suitable mixture of nutrients and growth factors. Cell culture is now a well established technique and is widely used for several purposes. For example, the responseof selectedanimal cells to the addition of chemical agents has led to a better understanding of hormone acon 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 counercially 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 public health during the last 40 years. 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.
il Plant tissue culture is normally used as a blanket term to cover the cultiation in controlled environmental condions n aitro of aLlplant parts whether single cell, a group of cells or an orgrn.Animal tissue culture involves tlre growth of cells in a tissue matrix. In tis chapteq cell culture is used to mean the grbw&of isolated cells.

Explant Coverslip

3.2 Thebeginnings of animaland plant cell culture

Several individuals were associatedwith the development of cell culture
biological : a commercially iseful proiluctfrom cells.lt may bea singlecompound or a complex mixture. ReferenceMorgan, S.I.and Darling, D.C. (1993)Animal CeIICulture,Biqs,Oxford, UK. This text describes practical details as well as the fundamentals associatedwith cell culture.

Fig.3.f An illustration of Harrison's hanging (1907). droptechnique Thiswas oneof the first methodsreported for growingeukaryotic cellsrn vitro.

Introduction 87

of biologicals in thecultureof animaland plant cells Table3.7 Milestones for production Animal cells Chick embryo cells maintained in vitro in saline Frog nerve cells cultivated using'hanging drop' Aseptic techniques introduced Continuous cell line from human cervical carcinoma cells established (HeLa cells) First widely used chemically defined medium (Eagle's) Human fibroblasts shown to have a finite life-span in culture 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 1883 1885 1900-1910 193U7940 1950-1960 Plant cells Cell theory proposed Totipotency postulated, attempts at plant cell culture Indoleaceticacid (plant hormone) isolated Study of rubber biosynthesis in guayule callus pioneered Auxin/cytokinin interaction found necessaryfor organogenesis Early efforts to grow cultures in millilitre suspensionsystems lnorganic growth medium investigated

L96Vr970

L970-1980

Continuous culture of cell suspensions Organ cultures developed that produced many different compounds 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 Development of immobilized cell systems Study of factors ifluencing expression gf secondary metabolites in cultures cells Hair-like root system transferred by Agrobocterium rhizogmes used as cell cultures in bioreactors for producing secon&ry metabolites : Thxol, an anti-cancercompound, was isolated from cell cultures of Taxus

Recombinant DNA technology allows large-scale production of biologicals fron\ genetically engineered cells

1987-1995

t987

1990 Nine animal cell culture products are licensed for human therapeutic use (seeTiable3.8). 1987-1993

Box3.1
production Vaccine

The original impetusfor the applcaton of cellculture techniques from a laboratory toalargeindustria|Sca|earosefromtheabi|itytogrowVrUSeSinanima|ce|ls 1949it was shown that the poliomyelitis viruscouldbe grown in cellculture. This findingestablished the basisfor the development for of a large-scale technology production. The.polio vaccine was produced for masshumanvaccinaton vaccine in the 1950s. Sincethat time a rangeof otherbiologicals synthesized from anmal application cellshavefoundcommercal and havebeenresponsible for muchof interestin modernanimal the scientific and financial cellbiotechnology.

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 as pressure of which is thesme osmoticpotential intracellular fluid.

Animal cell.culture 89

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 recognzedby 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 g e l e c t r o nm i c r o g r a p h s of ( a ) f i b r o b l a s t(s x 8 2 5 ) a n d ( b ) e p i t h e l i a l c e l( ls x 7 5 0 ) b o t hg r o w i n go n c o l l a g e n bovine fibres. Courtesy 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 aS l ciences, t h e M a n c h e s t e rM e t r o p o l i t a n U 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 d b 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 r A . 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 , University C 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 hf h y b r r d o m a c 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 . 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 aS c e l l )( x 1 7 6 4 1C l ciences, the Manchester Metropolitan Un i v e r s i t yU , 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
tissue fibroblast: a celltype foundin connectiue in associntion with collagen epithelial cell: a celltypederiued from epithelia, whichis a layercoaering an internalor external surface.

Reference Butler, M. and Dawson, M. (eds) (1992) CelI Culture Labfax, Bios, Oxford, UK. This is a rapid reference source of essential data for culturing cells

-t
A ni mal cel lcul ture 91

Table3.4 Animal cell linescommonly usedin culture Cell line BHK CHO HeLa IMR-90 L McCoy MDCK MRC-5 Origin 9yrur, baby hamster kidney Chinese hamster ovary Human cervical carcinoma Human embryonic lung Mouse connective tissue Mouse origin similar to L cells (Madin Darby) canine kidney Human embryonic lung Mouse myeloma Human lymphoblastoid (Burkitt's lymphoma) Mouse connective tissue Human embryonic lung Human amnion African green monkey Characteristics Fibroblasts,anchorage-dependent but can be induced into suspension Epithelial-like, suspensioncells Epithelial-like, suspensioncells Diploid fibroblasts, limited life-span Fibroblasts,suspensioncells Fibroblasts,anchorage-dependent Elrithelial-like, anchorage-dependent Diploid fibroblasts, limited life-span Lymphoblast-like, suspensioncells Lymphoblast-like, suspensioncells Fibroblasts,anchorage-dependent Diploid fibroblasts, limited life-span Epithelial-like, limited life-span Fibroblasts,anchorage-dependent The exponentialgrowth of cells can be describedby the equation:N = N0.2" or logN = logNo+ x.log2,where N = final cell number,No= initialcell number,x = ufrbr of generations growth. Cpnsidera of exponential t i s s u ee x p l a n t of 10 mm'containing 1 0 ' c e l l s .D e t e r m i n e the maximum theoretical cell yield from the growth of such cellsafter 50 generations. What would be the culturevolume necessaryto accommodate such growth?

Mrc-11
Namalwa

3T3 wI-38 WISH Vero

From Butler, M. (1988)Biotechnology: A Comprehensiae Treatise, 6b,249-31.

Animal cell growth in culture 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 inoculaon. The exponential growth phase is a period of continuous cell doubli^g.Animal cells normally exhibit a doubling time of between L5 and 25 hours. Growth continues in batch culture to L-2 x 1.06 cell cm-3,which is the typical cell density sustainableby presently available media. The stationary phase is a period after growth when there is no changein the culture cell density. The phase occurs when the nutrients have been depleted or inhibitory metabolites have accumulated in the culture. Cell death may occur in some cell lines by apoptosis, which is characterized by DNA fragmentation and the formation of characteristic blebs on the cell surface (Chapter 16). In culture these cells exhibit a rapid decreasein viable cell 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. 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 confluent culture is subcultured into two new cultures; that is, a split ratio of 2. In this case the generation number and passage number are the same. Otherwise, generation number - passagenumber X split ra|lLo / 2. 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 incubtion 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) talccn for a cell Wulation to doublein number. apoptosis: cellileathcausedby theactiaation of intracellular degrailntiae enzryes. This is akin to 'suicide a mechanism' Chapter16). for cells.(See (or passagenumber:thenumberof subcultures performed passages) after theoriginal isolationof thecells from aprimary source.

(a)

a -o)
(J -c

3
o .2

(b)
c o c

Time Fig.3.7 Growth shown by cells growing in (a) batch and (b) continuousculture. In batch culture a finite amount of nutrient medium is present and growth of cells ceaseswhen an essential nutrient is depleted (A, lag phase; B, exponential phase; C, stationaryphase).In continuous culture, cells are constantlysuppliedwith nutrientsbecauseof the inflow of fresh medium.

Animal cellculture 93

1x1os

1X108

1x107
f) I

tr
C) a q)

1x106

1x105

1x104

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. Cell suspension cultures are obtained from a friable callus (FiS.3.10)which can readily disperse to give single cells and/ or cell aggregateswhen agitated in liquid medium. 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 o 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).
G. (eds) ReferenceStafford, A. ur,a Wut "n, (1991)Plant celland tissueculture, Open University Press,Milton Keynes.A readable text included in the OU PressBiotechnology seriesand describing the basics of culture methods. plant cells of proliferating callus:an aggregation daneloped in culture.

beet,two months Fig.3.10 (a)Callus of sugar (x3.5).(b)Sugar from afterinitiation beetcells 7 daysold (x 190). Courtesy culture, suspension Department Dr J. McEntyre, of Biological Metropolitan theManchester Sciences. UK. University,

Plant cellculture 95

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 highyielding cultures. These problems have not yetbeen fully resolved. mutants can also be induced artificially by Higtr product-yielding ultraviolet irradiation or chemical mutagens, for example, N-methyl-N-nitroN-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 and screening together with refinements in the environmental conditions during culture. Such improvements have led, in some cases, to plant cell suspesion cultures producing as much, or more, product (weight for weight) than the whole plant. Table 3.5 summarizes the culture conditions which influence the accumulation of secondary metabolites in plant cells. metabolites in ofsecondary the accumulation influence which conditions ofculture Table 3.5 Summary plant cells
Conditions before culture . disease-freestock plants . use of whole plants which yield high quantities of the desired substance External culture conditions . light o temPerature . culture vessel agitation
Internal culture conditions . o . o components of the medium aeration and culture mixing culture medium pH cell maturity

gy, gy and Biotechnolo MolecularBiolo Chapter 8

ffi The yield of the red pigment shikonin from cultures of Lithospermum is L5 times greater (weight erithrorhizor for weight) than that from the whole plant. Shikonin, which has antibacterial, anti-inflammatory and antifumour properties in addition to being a valuable dye, was the first plant tissue culture product to be commercialized (in 1983inJapan).

Apart from accumulating secondary metabolites, plant cell-scan also be used tocatalyse enzymatically the conversion of organic materials to commercially important biochemicals. The conversion of part of a molecule_ using a Bacterial uld fungal biological system is called biotransformation. biotrnsformations 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.

Interesting reference Brodelius, P. and Pendersen, H. (1993) Increasing secondary metabolite production in plant-cell culture by redirecting transport. Trendsin Biotechnology ,ll, 30-36.

of animaland plant cellcultures 3,5 Thescale-up

means increasing the capacity of a culture so that a laboratory-based Scale-up 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 priniples determined in thesesystemscan be used by th9 animal or plant cell ultur biotechnologist. However, there are a number of distinct differences between animal, plant and microbial cells which lead to fundamental differencesin apprach to the large-scaleculture of thesecells (Thble3.6).
identical cells ogene:tically clone:a population derioed from an indiaidualcell. in genetic somaclonal oariation: theincrease place whenhigherplants aariabilitywhichtalces areculturedin vitro.
Reference Smith, R.H. (1992) Plant tissue and experimenfs,Academic culture: techniques Press, San Diego. This is a manual of practical details and exercises in plant cell culture suitable for undergraduate and graduate courses.

97 Thescale-up of cellcultures

Continuous feed lmmobilized ceils Air bubble Water jacket Ai (d)

Fiq.3.74 Dextran microcarriers covered witn monolayers (x 17bO). of mammalian fibroblasts

Reservoir

Packed spneres (c)

Fig' 3.73 Types of bioreactors.(a) Direct-driveslow-speed,stirred tank bioreactor. (b) 2 dm3 airlift 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 ) fluidized r e c i r c u l a t l n g b i o r e a c t o r . R 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 ) led P^riryiples of Plant Biotechnology: An lntroduction to Genetic Enginetering ptaiis. n 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. Aeratio and mixing ne_ed careful control, since concentrations of the[ases, oxygen and carbon dioxide, may affect biomass and product yield. Such a fnenter design_is being used commercially for the prouction of monoclonal antibodies (section 3.6) from hybridoma cells grwn 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. hir system has been used for the production of foot-nd-mouth disease vaccine frm animal cell cultures. THE FLUIDIZED BED REACTOR relies on liquid flow to maintain cells in Thescale-up of cellcultures9g/

ReferenceLubiniecki,A.S. (ed.)(1990) tnrgeScale Mammalian CellCultureTechnology, Mrcel 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
cr) F ) 1no

Totalvirus
micrograph Fig.3. f6 Electron of the poliovirus ( 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 . W , ithington CotterillP , u b l i cH e a l t hL a b o r a t o r y Hospital, M a n c h e s t e rU , K.

ci
c .F (o =
L

lu'

Table 3.7 Examplesof airnl aaccinesin common use Human Polio (Salk)* Polio (Sabin) Measles Mumps Rubella Yellow fever Rabies* Influenza

c C)

Veterinary
Foot-and-mouth disease* Marek's disease Newcastle disease Rinderpest Rabies Canine distemper Swine fever Blue tongue Fowl pox

.=

r E x t r a c e llla u
V US

4 10 6 B (h) Time afterinfection

4, ; , assembly; n,; s y n t h e s s3 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 o2 r e l e a s eo f v i r u s e s .

*Produced as inactivated viruses. All othes 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. DISEASE VACCINE is the most extensively used FOOT-AND-MOUTH 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 Thesafetyol deriued products fro* cellculture

for humantherapeutic use,strictsafety Beforeanyanimalcellproductis approved ln particula it must be shownthat the productis must be observed. standards of andal sodoesnot contai n any D N A .Theabsence fre eo f a n yv i ruscontami nati on a tumori geniagent c contami nati ng the product. agai nst D N A i s a s a feguard at a level< 1 pg cm-",which is allow DNAto be detected Present techniques lowerthanthe amountof DNA required to causea ordersof magnitude several single tu 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 Khler 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

M o u s ei m m u n i z e d

Spleen cells removeo

ffi;U'Jil,"/
, Fusion I ,-1.--, E:

Mvetoma

+
I

+
l-

lncubationin HAT selectsfor HGPRT+ (seeBox 3.4) Cellscloned Supernatant assayed

f o rM a b

Positi ve clones
S Elected

/\

\,^. <u \

c e l l propagation

ln vitro

, In ascrtes 'dt' ProPasation rruid or m i c e \htk

Fig. 3.20 Overview of the production and selectionof a monoclonalantibody-secreting ro t h a t s h o w n i n F i g . hybridoma c e l l l i n e( s i m i l a t guanine 3 . 5 ( d ) )H . G P R Th , ypoxanthine phosphoribosyl transferase; HAT,hypoxanth ine, 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 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 genetic A selectable marker

(h ypoxanthi ne guani ne phosphori bosyl transferase) genei s a TheHGPRT The productof the gene is a commonlyusedmarkerfor geneticselection. pathwaymechanism in the salvage transferase enzymewhich functions of nucleotide synthesis andwhich allowsthe nitrogen bases,hypoxanthine and guanine, to be converted to theircorresponding nucleotides. Anotherenzyme, (TK), is alsoimportant thymidinekinase in this salvage mechanism for the of thymidine to its nucleotide. The alternative mechanism conversion for nucleotide synthesis is by a de novopathway from simpleprecursors. Normally for nucleotide cellscan use eitherof thesepathways synthesis. Cellswith a defective HGPRT enzymecan be selected from cultureby treatment with 8-azaguanine. Thispurineanalogue is toxicto cellsif incorporated into DNA with HGPRT. However, HGPRTcellsare insensitive to the followingmetabolism toxic effects of 8-azaguanine. mediumHAT HGPRT* in cultureby usingthe selective cellscan be selected (hypoxanthine, inhibitor is a metabolic aminopterin andthymidine). Aminopterin pathway, which blocksthe de novonucleotide of hypoxanthine but the presence in cellswith a functional allowsnucleotide synthesis and thymidine to proceed to the HGPRT cellsbut HGPRT enzyme. Thusthe HATmediumis growth inhibitory growth promoting to the HGPRT. cells.
hybridoma: a cellline originatingfrom thefusion of two parentcellse.g.thefusionof a Blymphocyte cellgiaesriseto antibody-secreting anda myeloma hybridomas.

ReferenceMilstein, C. (1980)Monoclonal American,243(4),6G7 4. antibodies.Scientific The inventor of monoclonals tells the story.

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 dm3 batch cultures. Normal human diploid fibroblasts can be induced to produce B-interferon by the supplementation of the cultures with a synthetic double-stranded RNA (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 treatment is to increase the life span of the mRNA for interferon, and this consequently improves the specific productivity.

AcD

:f

rycHxi

tI

.=
J

lrme after removal of inducer (h)

Therapeutic compounds produced from animal cell cultures

There are numerous compounds produced by animal cell cultures other than antibodies and the interferons that can be used for human therapy. Table 3.8 lists some animal cell culture products that have been approved for human therapeutic use following clinical trials. cellcultures licensed products Table 3.8 Approaed fromanimal
Product Full name Lymphoblastoid interferon Anti-CD3 murine monoclonal antibody

Fiq.3.23 The superinduction of interferonin humandioloidfibroblasts. olC (polyinosine:cytosine) is a syntheticRNA,while CHx and AcD are the antibiotics cycloheximide and The antibioticsare actinomycinD, respectively. addedto inducethe synthesis of interferon. Redrawnfrom Friedman, R.M. (1981)lnterferons a Primer,Academic Press,NewYork, USA.

Application
Treatment of hairy-cell leukaemia Prevents tissue rejection in organ transplants Treatment of thrombosis following coronary disease Stimulation of red blood cell development in kidney failure Treatment of growth hormone deficiency (dwarfism) Vaccine to prevent hepatitis B infection Stimulation of white blood cell development following cytotoxic chemotherapy Treatment of haemophilia A Reduces mucus viscosiW in cystic fibrosis

19ft6 79f36 t987 1989 t989 t989 r991 t992 t993

IFN-a OKT3

t-PA EPo hGH HBsAg G-CSF FVIII DNase I

Tissue-typeplasminogen activator Erythropoietin Human growth hormone Hepatitis B surface antigen Granulocvte colonv stimulating factoi Factor 8 Deoxyribonuclease

Fiq.3.24 Photomicrograph of CHO cells grown on surfaceof glassculturebottle.(x145). courtesy Dr J.A. Wright, Instituteof Cell Biology,University of Manitoba, Canada.

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

Exercise 3
ActinomycinD inhibitsthe formation of mRNA whereascycloheximide inhibitsprotensynthesisfrom mRNA. With this information, attempt to explainthe mechanismof interferonproduction followingthe doubleantibiotic treatment.

Box3.6

trnterferon

(See a l s oB o x2 . 11 )

in the treatment and some may be usedtherapeutically of viraldiseases Interferon and their 14).Thereare severaltypes of interferons forms of sancer(Chapter properties Their and immunoregulatory activities. include antiviral, antiproliferative potential they havebeen although as therapeutic agentshasnot beenfullyrealized One of the valuable in the treatmentof a limitednumberof typesof cancers. involved interferon its extraction from activated methodsof obtaining original from humanblooddonors.However, this methodwas leucocytes obtained required of the compound for treatment. inadequate to supplythe largequantities

Animal cellproducts 105

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 required, under defined conditions and thereby giving consistent production. Table 3.9 shows some of the industrial uses of natural plant products. Market volumes and costs vary considerably for these products. For instance, 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, there is a much larger consumer market world-wide for quinine than for jasmine, at an approximate ratio of jasmine to quinine of 1:L00.There is also a large world market for codeine phosphate (an analgesic) and for digitoxin (a cardiotonic). Both of these have a consumer demand at about three times more than quinine. As indicated earlier (section 3.4), enzyme systems from cell cultures can be used in industrial biotransformations. One of the best-known examples of using plant cells on a large scale is that of the such a biotransformation cardiovascular steroid digitoxin obtained from foxglove (Digitalis lanata).The relies upon a single enzyme-catalysed 12required biotransformation hydroxylation (Fig. 3.26).

Look aroundyou at home, at work or in the street and list some of the names of productsin common use that are derivedfrom plants.

ffi There are a number of commerciallv important plant enzymes, includin! amylases, papain, bromelin, ficin and pectinases from cereals,papaya, pineapple, fig and citrus fruits, respectively. All find use in the food industry. For example, papain is used in meat tenderization.

Definethe terms primarymetabolite and secondary metabolite, as derived fromplants.

plantproducts andtheirindustrial Table ofnatural uses 3.9 Examples

Plant species Common name Pyrethrum jasmine Quinine Opium poppy Yam Quinine Foxglove Thorn apple Periwinkle Scientific name cinerariaefolium Chrysanthemum lasminium officinalis Cinchonaledgeriana Thaumat ococ cusdanielli somniferum Papaaer Dioscorea deltoidea ledgerinna Cinchona Digitalis lanata Daturastramonium roseus Catharanthus Pyrethrin Jasmine Quinine Thaumatin Codeine Diosgenin Quinine Digoxin Scopolamine Vincristine
Agrochemicals Insecticide Cosmetics Perfume Food and drink Bittering agent Non-nutritive sweetener Pharmaceuticals Analgesic Antiferlitv Antimalarial Cardiotonic Antihypersensitive Antileukaemic

Plant product

Industrial use

Plant cellproducts107

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