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Serum in mammalian cell culture: weighing the challenges of bioprocessing, ethics and animal welfare.


Serum is the clear portion of blood obtained after removing cells, platelets and clotting factors (Taber, 1999). Serum contains amino acids, proteins, growth factors, hormones, vitamins, inorganic substances, nutrients and metabolites. In mammalian cell culture, serum is often used as supplement to culture media (added in the range of 5-15% (v/v)) to promote and sustain cell growth as well as provide buffering and protection to cells (Freshney, 2005). Serum can be obtained commercially from manufacturers and they are sourced from a variety of species including bovine, chicken, caprine (goat), equine, human, ovine, porcine and rabbit as depicted in Table 1 (Invitrogen online)). Although serum is commonly used in mammalian cell culture research practice at lab scale, it is not a choice at large scale production since presence of serum often complicates the purification 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 from animals also has implications in haram and halal status of the final products. This paper is to review the benefits, disadvantages, limitations and challenges of using serum in the mammalian cell culture bioprocessing.

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 presence of growth-promoting components, specifically, 30-50% (v/v) bovine serum albumin van der Valk et al. (2004) and fetuin (serum protein abundant in fetus) Schinke et al. (1996). However, as depicted in Figure 1, production of FBS from bovine fetuses posed an ethical issue. The upstream processing is seen as very cruel and against animal welfare.


Typically, a nine month fetus would only provide approximately 550 ml raw FBS (Table 2). For a one year supply of raw FBS (500, 000 L) (Jochems et al. (2002), around 1 million of nine-month old fetuses are needed. As a byproduct of beef industry, FBS supply is complicated by health concern such as BSE (bovine spongiform encephalitis), FMD (foot and mouth disease) and rinderpest, to name a few (Invitrogen online).

Donor Horse Blood and Donor Horse Serum:

Serum from donor horse is also available in the market. For instance, TCS Biosciences Ltd., UK, (TCSBiosciences online) has pioneered processes that guarantee consistent and high quality donor horse serum. The collection of blood from horse donor was claimed to be stress-free.

Fish Serum:

Fish serum from surimi wash water processing line has also been reported as a potential substitute for serum in mammalian hybridoma cell culture Zakaria-Runkat et al. (2006). However, to this end, the fish serum still requires improvement to meet its more superior fetal bovine serum counterpart.

Fanous (2010) described the of use salmon blood as a component of microbial growth media. 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 et al. (2011) showed that locally made serum from halal slaughtered bovine is comparable to commercially available serum 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 maximum viable cell numbers of 8.0 x [10.sup.5] cells/mL were obtained when culture was supplemented with locally produced bovine serum as compared to commercial bovine serum (8.5 x [10.sup.5] cell/mL and 6.78 x [10.sup.5] cell/mL) for GIBCO and PAA respectively after four days.

Serum quality control test:

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 commercial purposes are listed in Table 3.

Serum-supplemented media (SSM) vs serum-free media (SFM):

Benefits, limitations and challenges:

Despite being acknowledged as a universal supplement with a myriad of growth promoting substances for culture media, content of serum remains to be undefined. This undefined nature and its inherent physiological variability (Freshney, 2005) 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 formulation which includes serum-free media, animal-free media, protein-free media, chemically defined media and chemically animal-free defined media.

Collectively, serum-free media (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, simplify regulatory documentation, provide consistent media performance and reduce downstream purification challenges (Thermo Scientific, 2011). 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. Comparison of price between SFM and SSM could be very subjective. For instance, use of SFM in research lab where volume 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.


Case study: IGF-1 as ingredient in serum-free media:

Serum alternatives such as growth factors and extracelullar matrices are often added into SFM formulation. For instance, insulin-like growth factor-1 (IGF-1) is considered a key growth factor in industrial cell cultures; their removal from media can reduce the cell growth-promoting activity of a culture by as much as 90% (Park et al., 2006).

To complement use of serum free media, research has been focused on the development of cell lines that are cabaple of autocrine growth of cells in culture, rendering the reduced cost (of not having to supply serum as sources of IGF) (Sunstrom et al. 1998 and Baserga and Ardmore, 1993). For instance, Sunstrom et al. (1998) developed Super- CHO cell line (from 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 manipulating medium composition (SSM) itself since protein, energy requirement and serum have been shown to affect IGF system leading to cell growth.


Serum 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 bioproducts 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.


Ahmad Nor, Y., J. Nuhu Jaafar, and M. Mel, 2011. Efficient performance of locally processed serum in Vero cell culture. Journal of Materials Science and Engineering, 5: 439-446.

Baserga, R. and P. Ardmore, 1993. Cell lines which constitutively express IGF-I and IGF-I R. USPatent #5262308.

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.

Freshney, R.I., 2005. Culture of Animal Cells: A Manual of Basic Technique (5th ed.), Wiley-Liss, Inc.

Jochems, C.E.A., J. van der Valk, F.R. Stafleu and B.A. Baumans, 2002. The use of fetal bovine serum: ethical or scientific problem. Altern. Lab. Anim., 30: 219-227.

Park, H., S. An, and T. Choe, 2006. Change of Insulin-like Growth Factor Gene Expression In Chinese Hamster Ovary Cells Cultured in Serum-free Media. Biotechnology and Bioprocess Engineering, 11: 319-324.

Schinke, T., C. Amendt, A. Trindl, O. Poschke, W. Muller-Esterl and W. Jahnen-Dechent, 1996. The serum protein [[alpha].sub.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.

Sunstrom, N.A., M. Baig, D.P. Sugyiono and P. Gray, 1998. Recombinant insulin-like growth factor-I (IGF-I) production in super-CHO results in expression of IGF-I receptor and IGF binding protein 3. Cytotechnology, 28: 91-99.

Taber, C.W., 1989. Taber's Cyclopedic Medical Dictionary. Philadelphia: F.A. Davis Company.6th Edition.

TCS Bioscience Co. Ltd. Donor horse blood and donor horse serum available from TCS Biosciences Ltd. [accessed on 18th March 2011].

Technical notes. GIBRO[R] Serum--it passes the test. [accessed on 18th March 2011].

Thermo Scientific, 2011. Thermo Scientific Hy-Clone Serum Free Media Maximize Cell Culture Production and Performance. Utah, USA. [accessed on 18th March 2011].

Van der Valk, J., D. Mellor, R. Brands, R. Fischer, F. Gruber, G. Gstraunthaler, R. Hellebrekers, J. Hyllner, F.H. Jonjker, P. Priote, M. Thalen and V. Baumans, 2004. The humane collection of fetal bovine serum and possibilities for serum-free cell and tissue culture. Toxicol. In Vitro, 1-12.

Zakaria-Runkat, F., W. Worawattanamateekul, O. Lawhavinit, 2006. Production of fish serum products as substitute for fetal bovine serum in hybridoma cell cultures from surimi industrial waste. Kasetsart J (Nat Sci), 40: 198-205.

(1,2) Yumi Zuhanis Has-Yun Hashim, (1,2) Maizirwan Mel, (1,2) Hamzah Mohd Salleh, (1) Yusilawati Ahmad Nor, (1) Siti Hajar Othman and (1) Wan Yusra Hannanah Wan Abdul Razak

(1) Institute for Halal Research and Training (INHART) Ground Floor, Block E0, Kulliyyah of Engineering, International Islamic University Malaysia, P.O. Box 10, 50728 Kuala Lumpur, Malaysia

(2) Bioprocess and Molecular Engineering Research Unit (BPMERU), Department of Biotechnology Engineering, Kulliyyah of Engineering, International Islamic University Malaysia, P.O. Box 10, 50728 Kuala Lumpur, Malaysia

Corresponding Author: Institute for Halal Research and Training (INHART), Ground Floor, Block E0, Kulliyyah of Engineering, International Islamic University Malaysia, P.O. Box 10, 50728 Kuala Lumpur, Malaysia.

Table 1: Types of serum in the market. From Invitrogen.

Product              Description

Newborn calf serum   Collected from calves typically 20 days old or

Bovine serum         Collected from prime cattle with the age range of
                     12-36 month, but usually 24 months or younger

Donor bovine serum   Collected from prime cattle with the age range of
                     12--36 month, but usually 24 months or younger

Donor horse serum    From donor herd

Chicken serum        Collected from healthy birds slaughtered for
                     human consumption

Product              Collected from donor herd

Lamb serum           Collected from animals younger than 1 year old

Porcine serum        Collected from animals younger than 1 year old

Rabbit serum         Collected from animals typically 6 months old or

Table 2: Age of bovine fetus and volume of raw FBS (Fetal Bovine
Serum) (Jochems et al. (2002))

Age of bovine fetus (months)    Raw FBS obtained (ml)

3                               150
6                               350
9                               550

Table 3: Quality control test routinely performed in
serum processing and manufacturing (Invitrogen online)

Cytotoxity assay
Electrophoretic pattern
Endotoxin (EU/ml)
Hemoglobin (mg/dl)
Sterility testing (bacteria/fungi)
Viral testing
Total protein
Vero performance assay
Hormone profile
Cytophatic agent
Hemadsorbing agent
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Article Details
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Title Annotation:Original Article
Author:Hashim, Yumi Zuhanis Has-Yun; Mel, Maizirwan; Salleh, Hamzah Mohd; Nor, Yusilawati Ahmad; Othman, Si
Publication:Advances in Natural and Applied Sciences
Article Type:Report
Geographic Code:9MALA
Date:May 1, 2012
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