Serum-Free Media

Although many cell lines are still propagated in medium supplemented with
serum, in many instances cultures may now be propagated in serum-free
media (Tables 10.1, 10.2). The need to (1) standardie media among
laboratories, (2) pro!ide specialied media for speci"c cell type, and (#)
eliminate !ariable natural products, led to the de!elopment of more comple$
media, such as %1&& of %organ et al. '1&(0), *%+, 10-- of .ar/er et al.
'1&(0), 1*T*10& '2!ans et al., 1&(-), 3aymouth4s %5 (0261 '1&(&),
1*T*1#( '2!ans 7 5ryant, 1&-(), and 5irch and .irt '1&01) for ,&2& mouse
"broblast cells, and 8am4s 910 '1&-#) and 912 '1&-() clonal growth media
for *hinese hamster o!ary (*8:) cells. ;erum-free media were also
de!eloped for 8e,a human cer!ical carcinoma cells '5la/er et al., 1&01<
8iguchi, 1&00). Although a degree of cell selectionmay ha!e been in!ol!ed in
the adaptation of continuous cell lines to serum-free conditions, the %*=5
series of media '8am 7 %c>eehan, 1&0?< see also Table 10.1), ;ato4s
=%2%6912-based media '5arnes 7 ;ato, 1&?0), and others based on +.%@
1-A0 '*arney et al., 1&?1< 5rower et al., 1&?-< see also Table 10.2),
demonstrated that serum could be reduced or omitted without apparent cell
selection if appropriate nutritional and hormonal modi"cations were made to
the media '5arnes et al., 1&?AaBd< *artwright 7 ;hah, 1&&A< %ather, 1&&?).
These also pro!ided selecti!e conditions for primary culture of particular cell
types. ;peci"c formulations (e.g., %*=5 110 '5ettger et al., 1&?1)) were
deri!ed to culture human "broblasts '8am, 1&?A), many normal and
neoplastic murine and human cells '5arnes 7 ;ato, 1&?0), lymphoblasts
'@sco!e 7 %elchers, 1& ?), and se!eral diCerent primary cultures '%ather 7
;ato, 1&0&a,b< ;undD!ist et al., 1&&1< Eupta et al., 1&&0< >een 7 +apson,
1&&(< Fonen et al., 1&&2) in the absence of serum, with, in se!eral cases,
some protein added 'Tsao et al., 1&?2< 5enders et al., 1&&1). This list now
co!ers a wide range of cell types, and many of the media are a!ailable
commercially (see Appendi$ @@). @n addition, the need to remo!e animal
proteins from the in !itro production of biopharmaceuticals has generated a
number of formulations for continuous cell lines such as *8: and
hybridomas '9roud, 1&&&< @/onomou et al., 200#< ;hah, 1&&&), not least for
some of the safety issues in!ol!ed '%erten, 1&&&).
10.1 =@;A=FA1TAE2; :9 ;2+G%
Gsing serum in a medium has a number of disad!antagesH
(1) Physiological Variability. The maIor constituents of serum, such as
albumin and transferrin, are /nown, but serum also contains a wide range of
minor components that may ha!e a considerable eCect on cell growth (see
Table &.(). These components include nutrients (amino acids, nucleosides,
sugars, etc.), peptide growth factors, hormones, minerals, and lipids, the
concentrations and actions of which ha!e not been fully determined.
(2) Shelf Life and Consistency. ;erum !aries from batch to batch, and at
best a batch will last one year, perhaps deteriorating during that time. @t
must then be replaced with another batch that may be selected as similar,
but will ne!er be identical, to the "rst batch.
(#) Quality Control. *hanging serum batches reDuires e$tensi!e testing to
ensure that the replacement is as close as possible to the pre!ious batch.
This can in!ol!e se!eral tests (for growth, plating eJciency, and special
functions< see ;ection &.0.2) and a number of diCerent cell lines.
(A) Specifcity. @f more than one cell type is used, each type may reDuire a
diCerent batch of serum, so that se!eral batches must be held on reser!e
simultaneously. *oculturing diCerent cell types will present an e!en greater
problem.
(() Availability. .eriodically, the supply of serum is restricted because of
drought in the cattle-rearing areas, the spread of disease among the cattle,
or economic or political reasons. This can create problems at any time,
restricting the amount of serum a!ailable and the number of batches to
choose from, but can be particularly acute at times of high demand. Today,
demand is increasing, and it will probably e$ceed supply unless the maIority
of commercial users are able to adopt serum-free media. Although an
a!erage research laboratory may reser!e 100B200 , of serum per year, a
commercial biotechnology laboratory can use that amount or more in a
wee/.
(-) Downstrea Processing. The presence of serum creates a maIor
obstacle to product puri"cation and may e!en limit the pharmaceutical
acceptance of the product.
(0) Containation. ;erum is freDuently contaminated with !iruses, many of
which may be harmless to cell culture but represent an additional un/nown
factor outside the operator4s control '%erten, 1&&&). 9ortunately,
impro!ements in serum steriliation techniDues ha!e !irtually eliminated the
ris/ of mycoplasma infection from sera from most reputable suppliers, but
!iral infection remains a problem, despite claims that some "lters may
remo!e !iruses (.all Eelman). 5ecause of the ris/ of spreading bo!ine
spongiform encephalitis among cattle, cell cultures and serum shipped to the
Gnited ;tates orAustralia reDuire information on the country of origin and the
batch number of the serum. ;erum deri!ed from cattle in 1ew Kealand
probably has the lowest endogenous !iral contamination, as many of the
!iruses found in 2uropean and 1orth American cattle are not found in 1ew
Kealand.
(?) Cost. *ost is often cited as a disad!antage of serum supplementation.
*ertainly, serum constitutes the maIor part of the cost of a bottle of medium
(more than 10 times the cost of the chemical constituents), but if it is
replaced by de"ned constituents, the cost of these may be as high as that of
the serum. 8owe!er, as the demand for such items as transferrin, selenium,
insulin, etc., rises, the cost is li/ely to come down with increasing mar/et
sie, and serum-free media will become relati!ely cheaper. The a!ailability of
recombinant growth factors, coupled with mar/et demand, may help to
reduce their intrinsic cost.
(&) !rowth "nhibitors. As well as its growth-promoting acti!ity, serum
contains growth-inhibiting acti!ity, and although stimulation usually
predominates, the net eCect of the serum is an unpredictable combination of
both inhibition and stimulation of growth. Although substances such as .=E9
may be mitogenic to "broblasts, other constituents of serum can be
cytostatic. 8ydrocortisone, present at around 1 L 10M? % in fetal serum, is
cytostatic to many cell types, such as glia 'Euner et al., 1&00) and lung
epithelium '%c,ean et al., 1&?-), at high cell densities (although it may be
mitogenic at low cell densities), and TE9-N, released from platelets, is
cytostatic to many epithelial cells.
(10) Standardi#ation. ;tandardiation of e$perimental and production
protocols is diJcult, both at diCerent times and among diCerent laboratories,
because of batch-tobatch !ariations in serum.
$%.& ADVA'(A!)S *+ S),-./+,)) .)D"A
All of the abo!e problems can be eliminated by remo!al of serum and other
animal products. ;erum-free media ha!e, in addition, two maIor positi!e
bene"ts.
$%.&.$ Selective .edia
:ne of the maIor ad!antages of the control o!er growth promoting acti!ity
aCorded by serum-free media is the ability to ma/e a medium selecti!e for
a particular cell type (see Tables 10.1 and 10.2). 9ibroblastic o!ergrowth
can be inhibited in breast and s/in cultures by using %*=5 100 '8ammond
et al., 1&?A) and 1(# '.eehl and 8am, 1&?0), melanocytes can be
culti!ated in the absence of "broblasts and /eratinocytes '1aeyaert et al.,
1&&1), and separate lineages and e!en stages of de!elopment may be
selected in hematopoietic cells by choosing the correct growth factor or
group of growth factors (see ;ection 2#.A). %any of these selecti!e media
are now a!ailable commercially along with cultures of selected cell types
(see Table 10.A).
$%.&.& ,egulation of Proliferation and Di0erentiation
Add to the ability to select for a speci"c cell type the possibility of switching
from a growth-enhancing medium for propagation to a diCerentiation-
inducing medium by altering the concentration and types of growth factors
and other inducers.
$%.1 D"SADVA'(A!)S *+ S),-./+,)) .)D"A
;erum-free media are not without disad!antagesH
2$3 .ultiplicity of .edia.
2ach cell type appears to reDuire a diCerent recipe, and cultures from
malignant tumors may !ary in reDuirements from tumor to tumor, e!en
within one class of tumors. Although this degree of speci"city may be an
ad!antage to those isolating speci"c cell types, it presents a problem for
laboratories maintaining cell lines of se!eral diCerent origins.
2&3 Selectivity.
Gnfortunately, the transition to serum-free conditions, howe!er desirable, is
not as straightforward as it seems. ;ome media may select a sublineage
that is not typical of the whole population, and e!en in continuous cell
lines, some degree of selection may still be reDuired. *ells at diCerent
stages of de!elopment (e.g., stem cells !s. committed precursor cells) may
reDuire diCerent formulations, particularly in the growth factor and cyto/ine
components.
213 ,eagent Purity.
The remo!al of serum also reDuires that the degree of purity of reagents
and water and the degree of cleanliness of all apparatus be e$tremely high,
as the remo!al of serum also remo!es the protecti!e, deto$ifying action
that some serum proteins may ha!e. Although remo!ing this action is no
doubt desirable, it may not always be achie!able, depending on resources.
243 Cell Proliferation.
Erowth is often slower in serumfree media, and fewer generations are
achie!ed with "nite cell lines.
253 Availability.
Although impro!ing steadily, the a!ailability of properly Duality-controlled
serum-free media is Duite limited, and the products are often more
e$pensi!e than con!entional media.