Download Document

2nd International Conference on Professional Ethics and Education in Engineering 2011 ( ICEPEE’11)
Serum In Mammalian Cell Culture: Weighing The
Challenges of Bioprocessing, Ethics and Animal
Welfare
Yumi Zuhanis Has-Yun Hashim, Maizirwan Mel, Hamzah Mohd Salleh, Yusilawati Ahmad Nor, Siti Hajar
Othman and Wan Yusra Hannanah Wan Abdul Razak
Bioprocess and Molecular Engineering Research Unit (BPM ERU),
Depart ment of Biotechnology Engineering,
Kulliyyah of Engineering
International Islamic University Malaysia
Kuala Lu mpur, Malaysia
yumi@iiu m.edu.my
Abstract— S erum; a clear portion of blood obtained after
removing cells, platelets and clotting factors is a universal
supplement commonly used in media for mammalian cell culture
system. Serum contains amino acids, proteins, growth factors,
hormones, vitamins, inorganic substances, nutrients and
metabolites; which promote and sustain cell growth as well as
provide buffering condition and protection to cells. Serum, from
various animal sources such as bovine and porcine can be
obtained commercially from manufacturers. Although serum has
long been used successfully in mammalian cell culture system,
recent trend is moving towards serum alternatives and serumfree media. This is due to many factors including the ethical and
animal welfare issues in serum processing and production.
Further, large scale production of bio-products using mammalian
cell culture system prefers defined media which offers
reproducibility and ease of downstream purification.
Nevertheless, development of serum-free media is tedious and
somewhat involve high cost with inherent problem of low growth
rate as compared to serum supplemented media. This review is
set to explore the many facets of serum and serum-free media in
mammalian cell culture bioprocessing.
variety of species including bovine, chicken, caprine (goat),
equine, human, ovine, porcine and rabbit as depicted in Figure
1 [3]. Although serum is common ly used in mammalian cell
culture research practice at lab scale, it is not a choice at large
scale production since presence of serum often co mplicates
the purificat ion of bio-products. Further, recent trend is also
moving towards use of serum-free media or serum alternatives
due to animal welfare and ethical issues in serum processing
and production. Further, use of serum fro m animals also has
implications in haram and halal status of the final products.
This paper is to rev iew the benefits, d isadvantages, limitations
and challenges of using serum in the mammalian cell cu lture
bioprocessing.
TABLE I TYPES OF SERUM IN THE MARKET.
FROM [3].
Keywords- Serum, serum-free media, ethical issues, mammalian
cell culture, bioprocessing
INT RODUCTION
Seru m is the clear portion of blood obtained after
removing cells, platelets and clotting factors [1]. Seru m
contains amino acids, proteins, growth factors, hormones,
vitamins, inorganic substances, nutrients and metabolites. In
mammalian cell culture, seru m is often used as supplement to
culture med ia (added in the range of 5 – 15 % (v/v)) to
promote and sustain cell gro wth as well as provide buffering
and protection to cells [2].
Seru m can be obtained
commercially fro m manufacturers and they are sourced fro m a
V.
PRODUCTION OF SERUM
Fetal Bovine Serum
Among the many varieties, Fetal bovine serum (FBS) is
the serum of choice in mammalian cell culture due to the
ISBN : 978-983-42978-2-4
338
2nd International Conference on Professional Ethics and Education in Engineering 2011 ( ICEPEE’11)
presence of growth-pro moting co mponents, specifically, 30 –
50 % (v/v) bovine seru m albu min [4] and fetuin (serum
protein abundant in fetus) [5]. However, as depicted in Figure
1, production of FBS fro m bovine fetuses posed an ethical
issue. The upstream processing is seen as very cruel and
against animal welfare. Typically, a n ine month fetus would
only provide approximately 550 ml raw FBS (Tab le 1). For a
one year supply of raw FBS (500, 000 L) [6], around 1 million
of nine-month old fetuses are needed. As a byproduct of beef
industry, FBS supply is co mplicated by health concern such as
BSE (bovine spongiform encephalitis), FMD (foot and mouth
disease) and rinderpest, to name a few [3].
Slaughter of
pregnant cow
Downstream
• Reproductive tract with fetus removed to
processing area
• Calf removed, cleaned and disinfected
• Blood collection (heart puncture/umbilical cord
puncture)
• Blood clotting, serum separation
• Raw serum tested for Quality Control
• Refrigerated centrifugation
• Filtration (0.22 m filter)
• UV radiation
• Heat activation at 56C
• Packaging
• Frozen for distribution
mammalian hybridoma cell cu lture [8]. However, to this end,
the fish serum still requires imp rovement to meet its more
superior fetal bovine serum counterpart.
Fanous [9] described the of use salmon blood as a
component of microbial gro wth med ia. It contains easily
digested proteins and a high concentration of poly-unsaturated
omega-3 acids.
The use of salmon blood thus can be
extended to mammalian cell culture. Nevertheless, the halal
status of fish blood (serum) is still being debated.
Locally Produced Bovine Serum
Ahmad Nor [10] showed that locally made serum fro m
halal slaughtered bovine is comparable to co mmercially
available seru m used in Vero cell culture. The blood was
collected at slaughtering and processed according to standard
procedure for serum processing. The author reported that the
maximu m viable cell numbers of 8.0 × 105 cells/ mL were
obtained when culture was supplemented with locally
produced bovine serum as compared to co mmercial bovine
serum (8.5 × 105 cell/ mL and 6.78 × 105 cell/ mL) for GIBCO
and PAA respectively after four days.
SERUM QUALITY CONT ROL TEST
Figure 8.
Processing of serum (Fetal Bovine Seru m,
FBS) Adapted from [5].
TABLE II.
AGE OF BOVINE FETUS AND VOLUME OF RAW
FBS (FET AL BOVINE SERUM )[6]
Age of bovine
fetus (months)
Raw
FBS
obtained (ml)
3
150
6
350
9
550
Production of serum involves strict quality control tests
which ensure quality products but at the same time lend to its
high price in the market. Several quality control tests routinely
performed during processing of serum for co mmercial
purposes are listed in Table III.
TABLE III. QUA LITY CONTROL TEST ROUTINELY
PERFORM ED IN SERUM PROCESSING
A ND
MANUFA CTURING[3].
Donor Horse Blood and Donor Horse Serum
Seru m fro m donor horse is also available in the market.
For instance, TCS Biosciences Ltd., UK, [7] has pioneered
processes that guarantee consistent and high quality donor
horse serum. The co llection of blood fro m horse donor was
claimed to be stress-free.
Fish Serum
Fish serum fro m surimi wash water processing line has
also been reported as a potential substitute for serum in
ISBN : 978-983-42978-2-4
339
2nd International Conference on Professional Ethics and Education in Engineering 2011 ( ICEPEE’11)
SERUM-SUPPLEM ENTED M EDIA (SSM ) VS SERUM FREE M EDIA (SFM)
Benefits, limitations and challenges
Despite being acknowledged as a universal supplement
with a myriad of growth promoting s ubstances for culture
med ia, content of serum remains to be undefined. This
undefined nature and its inherent physiological variability [2]
appear to be a major obstacle to the production of
biopharmaceuticals in animal cell culture system. Other
technical disadvantages of serum include risk of
contamination, ethical issues, market condition and animal
supply leading to fluctuating price. This has spurred interest to
develop a more defined formu lation wh ich includes serumfree med ia, animal-free media, protein-free media, chemically
defined media and chemically animal-free defined med ia.
Collectively,
serum-free med ia (SFM) has more
advantages as compared to the serum-supplemented media
(SSM ) (Figure 2). SFM has been claimed to enable
elimination of pre-screen serum lot, simp lify regulatory
documentation, provide consistent media performance and
reduce downstream purification challenges [11]. However,
SFM is not without flaws where cell growth is often slower in
this type of media. SFM could also be very specific to certain
cell types at certain growth phase hence the need to have very
selective media leading to increased cost. Co mparison of price
between SFM and SSM could be very subjective. For instance,
use of SFM in research lab where volu me is not that high
would be considered expensive while use of SFM in large
scale manufacturing of bioproducts may be cheap in terms of
overall cost and return of investment.
Seru m alternatives such as growth factors and extracelullar
matrices are often added into SFM formu lation. For instance,
insulin-like growth factor-1 (IGF-1) is considered a key
growth factor in industrial cell cultures; their removal fro m
med ia can reduce the cell gro wth-pro moting activity of a
culture by as much as 90% [12].
To complement use of serum free media, research has
been focused on the development of cell lines that are cabaple
of autocrine growth o f cells in cu lture, rendering the reduced
cost (of not having to supply serum as sources of IGF) [13,
14]. For instance, Sunstrom et al [13] developed Super- CHO
cell line (fro m Chinese Hamster Ovary (CHO) parent cell
line) which expressed recombinant IGF-I that renders the cells
to grow indifinitely in protein and/or serum-free media.
Although this looks promising, the absence of serum generally
will impose other issues such as induction of phenotypic
change due to changes at gene expression level. Other
approach includes regulation of IGF(s) by man ipulating
med iu m co mposition (SSM) itself since protein, energy
requirement and serum have been shown to affect IGF system
leading to cell growth.
CONCLUSION
Seru m has been traditionally and successfully used in
the mammalian cell culture system. However, current issues
pertaining the ethical, animal welfare and to some extent; the
halal and haram status; has directed new developments of
serum alternatives and SFM. The latter would also benefit the
large scale production of b io-products from mammalian cell
culture system. Nevertheless, one needs to carefully weigh the
benefits and disadvantages of SSM and SFM based on
informed choices to meet the desired outcome.
REFERENCES
[1]
[2]
[3]
[4]
[5]
Figure 2 Co mparison of seru m supplemented media (SSM)
and
serum-free media (SFM ) particularly in large
scale manufacturing of b ioproducts. DSP: downstream
processing.
[6]
[7]
Case study: IGF-1 as ingredient in serum-free media
[8]
ISBN : 978-983-42978-2-4
340
Taber, C.W. (1989). T aber's Cyclopedic Medical Dictionary.
Philadelphia: F.A. Davis Company.6th Edition.
Freshney, R.I., (2005). Culture of Animal Cells: A Manual of Basic
Technique (5 th ed.), Wiley-Liss, Inc.
T echnical notes. GIBRO® Serum – it passes the test.
http://www.invitrogen.com
van der Valk, J.; Mellor, D.; Brands, R.; Fischer, R.; Gruber, F.;
Gstraunthaler, G.; Hellebrekers, R.; Hyllner, J.; Jonker, F.H.; Priote, P.;
Thalen, M.; and Baumans, V. (2004). The humane collection of fetal
bovine serum and possibilities for serum-free cell and tissue culture.
Toxicol. In Vitro. 1-12.
Schinke, T.; Amendt, C.; Trindl, A.; Poschke, O.; Muller-Esterl, W. and
Jahnen-Dechent, W. (1996). The serum protein
2 -HS
glycoprotein/fetuin inhibits apatite formation in vitro and in mineralizing
calvaria cells. A possible role in mineralization and calcium
homeostasis. J. Biol.Chem. 271(34). 20789-20796.
Jochems, C.E.A.; van der Valk, J.; Stafleu, F.R. and Baumans, B.A.
(2002). The use of fetal bovine serum: ethical or scientific problem.
Altern. Lab. Anim. 30: 219-227.
T CS Bioscience Co. Ltd. Donor horse blood and donor horse serum
available from TCS Biosciences Ltd. http://www.t csbiosciences.co.uk.
Retrieved 18 th March 2011.
Zakaria-Runkat, F., Worawattanamateekul, W. and Lawhavinit, O.
(2006). Production of fish serum products as substitute for fetal bovine
2nd International Conference on Professional Ethics and Education in Engineering 2011 ( ICEPEE’11)
[9]
[10]
[11]
[12]
[13]
[14]
serum in hybridoma cell cultures from surimi industrial waste. Kasetsart
J (Nat Sci). 40 (Suppl):198-205.
Fanous, A. (2010). Growth media for enzymes and starter culture in
Halal perspective. World Halal Research Summit. Inspiring Innovation
through Halal Research. 23 – 25th June 2010. Kuala Lumpur.
Ahmad Nor, Y; Nuhu Jaafar, N. and Mel, M. (2011). Efficient
performance of locally processed serum in Vero cell culture. Journal of
Materials Science and Engineering. In press.
Thermo Scientific. Thermo Scientific Hy-Clone Serum Free Media
Maximize Cell Culture Production and Performance. Utah, USA.
Park, H., An, S. and Choe, T., (2006), Change of Insulin-like Growth
Factor Gene Expression In Chinese Hamster Ovary Cells Cultured in
Serum-free Media. Biotechnology and Bioprocess Engineering. 11: 319324.
Sunstrom, N.A.; Baig, M.; Sugyiono, D.P. and Gray, P. (1998).
Recombinant insulin-like growth factor-I (IGF-I) production in superCHO results in expression of IGF-I receptor and IGF binding protein 3.
Cytotechnology. 28: 91-99.
Baserga, R. and Ardmore, P. (1993). Cell lines which constitutively
express IGF-I and IGF-I R. USPatent #5262308.
ISBN : 978-983-42978-2-4
341